| Background and objectiveThe reconstruction of horizontal bony defects remains a challenging clinical problem in the case of periodontal supporting tissues lost due to inflammation, trauma, and tumors. Great progress in bone tissue engineering has been seen in recent years, however, various basic problems have to be solved before clinical application. There is still a large gap between tissue-engineered bones and the natural physiological bones. The main reasons is probably no engineered bone to achieve the synchronization of vascularization and neurotization.Growth factors play important roles in bone forming through stimulating the differentiation of mesenchymal stem cells into hard-tissue forming cells. Over the last few years, numerous growth factors, including fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF) and bone morphogenic proteins (BMPs), have been examined for their bone regenerative potential. Among these, recombinant human bone morphogenetic protein-2(rhBMP2) is known as one of the most potent growth factors that promote periodontal regeneration. Besides the encouraging results for the amount of regenerated bone, rhBMP-2could be used safely in human patients, and areas treated with rhBMP-2displayed normal bone formation. On the other hand, several studies in humans and animals showed that the quality of regenerated bone following the application rhBMP-2was compromised. The major drawbacks of regenerated bone induced by rhBMP-2are low clinical union rate as well as bone healing rate, weak mechanical stability, and ankylosis. These abnormalities are barriers to its clinical use for the repair of large bone defects. Unfortunately, limited work has been performed to improve the quality of regenerated bone. It is therefore necessary to find novel treatments that could improve not only the quantity but also the quality of regenerated bone.Recently, there is growing evidence that the nervous system is closely related to bone formation, repair and remodeling. Neuropeptide-containing nerves are widely distributed in normal bone tissue, especially in newly formed bone tissue. Moreover, patients with nerve injuries exhibit increased fracture rate and ectopic ossification, and kittens with nerve lesions show reduced bone quantity and quality. Additionally, in experimentally denervated rats, changes in fracture healing and weaker fracture calluses were observed, and in inferior alveolar nerve-transected rats, alveolar bone remodeling activity was inhibited. RhBMP-2-induced bone exhibited similar characteristics (ectopic ossification, weaker fracture calluses, and inhibited bone remodeling activity) to those in patients or experimental animals with neurological disorders. We hypothesized that neurotrophic growth factors, important regulators of the nervous system, may also influence bone remodeling as well.Nerve growth factor (NGF), a neurotrophin that plays a key role in promoting the growth and differentiation of sympathetic and sensory neurons, has recently been reported to enhance the bone remodeling activity, and play an important role in the healing processes of fractured bone. Furthermore, NGF increases expressions of bone/cementum-related proteins, osteopontin and alkaline phosphatase in human periodontal ligament cells and modulates the proliferation and differentiation of endothelial cells and periodontal ligament cells in vitro, which suggests its potential for periodontal tissue regeneration in vivo.Thus, in this study, rhBMP2and rhβ-NGF were locally applied to surgically prepared furcation class III defects in beagle dogs and the quality and quantity of regenerated bone in periodontal healing were investigated. The morphology and distribution of neural elements in the regenerated periodontal ligament were also evaluated. MethodsPart â… :Thirty-six inflamed furcation class â…¢ defects were created in six beagle dogs at sites of mandibular premolar2,3and4, and then biodegradable hydrogel incorporating rhBMP2and rhβ-NGF was topically applied to the defects. The groupings were as follows: G1:untreated (control group A), G2:carrier alone (control group B), G3:0.4%rhBMP2+carrier, G4:2%rhβ-NGF+carrier, G5:0.4%rhBMP2+2%rhβ-NGF+carrier, and G6:0.2%rhBMP2+1%rhβ-NGF+carrier. Eight weeks after application, the quality and quantity of regenerated tissue were evaluated by scanning electron microscopy (SEM) observation, calcium/phosphorus (Ca/P) ratio analysis, and histological evaluation.Part â…¡:To investigate the morphology and distribution of neural elements in the regenerated periodontal ligament (PDL),36class â…¢ furcation defects (5mm high) were surgically created in6beagle dogs at the location of mandibular premolar2,3and4. Following a healing period of8weeks, the regenerated neural elements in the PDL were observed by immunohistochemistry with protein gene product9.5(PGP9.5) antibody.ResultsPart!:rhBMP2alone could enhance the quantity of regenerated bone. A higher Ca/P ratio and a greater amount of newly formed newly formed alveolar bone were observed in rhp-NGF alone group than the two control groups. Moreover, the SEM observation revealed that the regenerated tissue in rhβ-NGF alone group showed larger diameter of collagen bundles than control groups. The dual release groups of rhBMP2and rhβ-NGF showed higher Ca/P ratios compared to the2control groups, with more calcifications on the collagen fiber surface. Moreover, the co-application of rhBMP2and rhβ-NGF stimulated larger area of regenerated bone and longer newly formed cementum than either the rhBMP2or rhp-NGF alone group, which indicates a synergistic effect of the two growth factors on periodontal regeneration. And our data suggest that the combination of the two growth factors improved the quality and quantity of regenerated bone in a dose-dependent manner.Part II:Our results showed that PGP9.5-immunoreactive fibers were sparsely found in all regenerated PDLs of class â…¢ furcation defects created in beagle dogs. Free nerve endings were predominant in the regenerated neural elements. Ruffini-like endings were observed only in3out of17specimens, and they showed less dendritic ramifications and branches. The percentage of the PGP9.5-immunopositive area (POP) in the notch-region of regenerated PDL was1.93±0.92%(mean±S.E.), which is significantly lower than the POP in the normal PDL (3.09±1.12%). The majority of the PGP9.5-positive fibers were observed adjacent to blood vessels and some of them ran along periodontal collagen fibers. Occasionally, the sprouting of nerve fibers was observed in the remodeling areas where periodontal tissue regeneration was in process.ConclusionsThe results of this pilot study suggest that a topical application of rhBMP2and rhβ-NGF may improve the quality and quantity of regenerated bone in artificially created furcation class â…¢ defects of beagle dogs. Ruffini-like endings can be rarely formed in the regenerated PDL of class â…¢ furcation defects created in beagle dogs, and neural elements may be involved in the periodontal tissue remodeling. |
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