BDNF localizes in the injured IAN.

Histological analysis of inferior alveolar nerve (IAN) injury experiments in rats after 14 days. Azan staining (A, B) and immunohistochemical stainings of BDNF (C-G, I) and trkB (H, J). (A, C, E, G, H) Unaffected region of IAN injury. (B, D, F, I, J) Regenerating IAN region after injury. Although unaffected IAN and peripheral bone tissues did not express BDNF (C, E, G), injured IAN (D, F) and active osteoblasts on the surface of newly formed bone (I) were immunopositive for BDNF. The osteoblast lineage cells surrounding newly formed bone were immunopositive for trkB (J). IAN; inferior alveolar nerve. Bars, 100 μm (A-F), 20 μm (G-J).</p

Local BDNF promotes bone formation after osteotomy in the mandible.

Micro-computed tomography images in the osteotomy at day 28 in rat. (A) The area of osteotomy was indicated by arrow (A). Twenty-eight days after osteotomy, exogenous BDNF accelerated regeneration of the cortex and induced osteosclerotic changes in the surrounding cortical bones (A). (B) Buccal cortical bone areas around osteotomy injury within 1,800 μm2 were measured using graphics software. The bone area was significantly wider in BDNF treated group.</p

Locally Produced BDNF Promotes Sclerotic Change in Alveolar Bone after Nerve Injury

<div><p>Brain-derived neurotrophic factor (BDNF), which is released due to nerve injury, is known to promote the natural healing of injured nerves. It is often observed that damage of mandibular canal induces local sclerotic changes in alveolar bone. We reported that peripheral nerve injury promotes the local production of BDNF; therefore, it was possible to hypothesize that peripheral nerve injury affects sclerotic changes in the alveolar bone. This study aimed to evaluate the effect of BDNF on osteogenesis using <i>in vitro</i> osteoblast-lineage cell culture and an <i>in vivo</i> rat osteotomy model. MC3T3-E1 cells were cultured with BDNF and were examined for cell proliferative activity, chemotaxis and mRNA expression levels of osteoblast differentiation markers. For <i>in vivo</i> study, inferior alveolar nerve (IAN) injury experiments and mandibular cortical osteotomy were performed using a rat model. In the osteotomy model, exogenous BDNF was applied to bone surfaces after corticotomy of the mandible, and we morphologically analyzed the new bone formation. As a result, mRNA expression of osteoblast differentiation marker, osteocalcin, was significantly increased by BDNF, although cell proliferation and migration were not affected. In the <i>in vivo</i> study, osteopontin-positive new bone formation was significantly accelerated in the BDNF-grafted groups, and active bone remodeling, involving trkB-positive osteoblasts and osteocytes, continued after 28 days. In conclusion, BDNF stimulated the differentiation of MC3T3-E1 cells and it promoted new bone formation and maturation. These results suggested that local BDNF produced by peripheral nerve injury contributes to accelerating sclerotic changes in the alveolar bone.</p></div

Strong expression of trkB in osteoblasts and osteocytes in response to exogenous BDNF.

<p>Immunohistochemical staining of BDNF at day 14 and day 28 (A) and trkB at day 7, day 14 and day 28 (B) after osteotomy. BDNF was deposited within newly formed bone matrices at day 14 and day 28 in the BDNF-treated group (A, arrows). TrkB-positive osteocytes were few in the control group (B). On the other hand, the strongly positive immunoreaction of trkB was observed in the osteoblasts (arrowheads) and osteocytes in bone surface and subsurface area (arrows) at day 7, day 14 and day 28 in the BDNF-treated bones (B). The number of trkB-positive osteocytes in the bone surface area was significantly higher in BDNF group at day 7 (C). Bars, 50 μm.</p

Local BDNF induces osteosclerotic changes to surrounding bones.

<p>Hematoxylin and eosin staining (A-L) and azan staining (K, L) on day 7 (A-D), day 14 (E-H) and day 28 (I-L) after osteotomy in rat. (C, D, G, H, K, L) Magnified views of square areas in A, B, E, F, I, J. Histological examination demonstrated that many resorption lacunae were observed on the bone surface at day 7 in the control group (C, arrow and square). In contrast, the cortical bone surface was smooth with few resorption lacunae in BDNF group (D). After day 14, active osteoblasts lined the surface of the BDNF-treated bone (H, arrow and square), although osteoblast differentiation was not prominent in control (G). At day 28, a number of complicated reversal lines were observed in the BDNF-treated bone, and active bone formation occurred not only on the outer surface of the cortical bone but also inside of the bone lacunae and bone marrow cavity as clearly demonstrated by azan staining (L, arrow and square). Bars, 1000 μm (A, B, E, F, I, J), 100 μm (C, D, G, H, K, L).</p

Sclerotic changes in alveolar bone after IAN injury.

<p>CT, X-ray and photographic images from patients of a post-extraction tooth cavity. (A) A thirty-five-year-old woman reported dysesthesia for a long period after the tooth extraction. Sclerotic changes were observed in the periphery of the alveolar bone (arrows). (B) A 46-year-old woman experienced dysesthesia, which increased in relation to the deformity of the mandibular canal. In both of these patients, the masses of traumatic neuroma located in the nerve lesion were detected later by surgical observation.</p

Prolonged active bone remodeling due to local BDNF.

Immunohistochemical staining of cathepsin K, ALP and osteopontin at day 7 (A) and day 28 (B) after osteotomy. Many cathepsin K-positive osteoclasts appeared in the control group at day 7. In contrast, osteopontin-positive lines were prominent in the BDNF-treated bone surface (A, C). At day 28, cathepsin K-positive osteoclasts were scattered on the surface of newly formed bone and bone lacunae in the BDNF group. In addition, osteoblast-lineage cells were strongly immunopositive for ALP, and OPN-positive new bone matrix (arrows) was significantly thicker in the BDNF group (B, C). Bars, 50 μm.</p

BDNF did not affect cell proliferation and migration in MC3T3-E1 cells.

<p>MC3T3-E1 cells were treated with recombinant BDNF. (A) Cell proliferation assay. BDNF did not affect the proliferation of MC3T3-E1 cells. (B) Cell migration assay. BDNF did not promote cell movement according to <i>in vitro</i> scratch assay.</p

BDNF promotes activation of Akt signals in MC3T3-E1 cells.

<p>Western blot analysis (A) and immunocytochemical staining of Akt and pAkt (Ser473) (B) in MC3T3-E1 cells. (A) BDNF significantly increased the active form of Akt (pAkt) in MC3T3-E1 cells at 30 min. On the other hand, the phosphorylation of ERK was not affected by BDNF. (B) BDNF enhanced pAkt immunoreactivity in the cytoplasm. The nucleus is stained with DAPI (blue).</p

Graphic summary of the predicted mechanism of bone sclerosis after IAN.

<p>Peripheral nerve injury was caused by surgical maneuver, e.g., tooth extraction, and it invaded the inferior alveolar nerve, inducing local diffusion of BDNF at the lesion. BDNF stimulated the differentiation of osteoblasts through trkB receptor, resulting in sclerotic changes in the bone. As a result, osteogenesis and remodeling over the bone cavity wall were facilitated, making the wall thicker and resulting in isolation of the surrounding alveolar tissue. OB, osteoblast; OS, osteocyte; OC, osteoclast; IAN, inferior alveolar nerve.</p