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Effects of Locally Applied Nerve Growth Factor to Mandibular Distraction Osteogenesis in Rabbits

Author: WangLei
Tutor: ZhouShuXia;LiuBaoLin;LeiDeLin
School: Fourth Military Medical University
Course: Clinical Stomatology
Keywords: Nerve growth factor Distraction osteogenesis Mandible Rabbit Inferior alveolar nerve Osteoblast Nano-HA/collagen composite Sustained release Bone repair Nerve regeneration
CLC: R782
Type: PhD thesis
Year: 2007
Downloads: 222
Quote: 1
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Abstract


Distraction osteogenesis (DO) has become a widely accepted method in craniofacial surgery and been applied to resolve clinical problems such as deformities and defects of mandible. However, the rather long period of bone consolidation time and inferior alveolar nerve (IAN) injury are two concerns in mandibular DO, and there are increasing demands for acceleration of bone maturation and enhancement of inferior alveolar nerve (IAN) recovery. Nerve growth factor (NGF) is an important growth factor involved in recovery of neurological injuries. On the other hand, NGF is also involved in normal bone metabolism and can enhance bone repair. So the main aim of this study was to determine the ability of locally applied NGF to enhance bone regeneration and recovery of IAN injury simultaneously in a rabbit model of mandibular DO. Six parts were included in this study:1 Effects of exogenous hNGFβon cultured rat osteoblastsObjective: To evaluate the effects of exogenous hNGFβon proliferation and osteoblastic ability of cultured rat osteoblasts. Methods: After rat osteoblasts were co-cultured with hNGFβat different concentrations, the cells’proliferation status (MTT assay), ALP activity, total protein content, calcium nodules formation, and immunostaining of typeⅠcollagen and osteocalcin were evaluated. Results: Data showed that proliferation of rat osteoblasts was not significantly affected by hNGFβ. ALP activity, total protein content, and expression of typeⅠcollagen and osteocalcin were significantly elevated by hNGFβat concentrations near 50 ng/ml in a dose-dependent manner. Conclusion: Exogenous hNGFβcan enhance osteoblastic ability of cultured rat osteoblasts without affecting their proliferation.2 hNGFβdelivery via a gel and its effects on cultured rat osteoblastsObjective: To fabricate a collagen/nano-hydroxyapatite/ k-carrageenan gel and determine the pattern of hNGFβsustaining release. To evaluate the effects of this gel on osteoblastic ability of cultured rat osteoblasts. Methods: Col/nHA, a bone-resembling material developed by Tsinghua University, was combined with k-carrageenan and 0.1 mg /ml hNGFβto fabricate a gel, and hNGFβsustaining release was evaluated in vitro. After co-cultured with this gel in a Transwell chamber, rat osteoblasts’proliferation status, ALP activity and total protein content were evaluated. Results: Release pattern of hNGFβfrom this gel indicated that erosion mechanism was included. ALP activity and total protein content were significantly enhanced by this gel/hNGFβcomposite. Conclusion: Col/nHA/k-carrageenan gel can be used as a good composite for hNGFβsustaining release. This gel/hNGFβcomposite can enhance osteoblastic ability of distantly cultured rat osteoblasts. 3 Effects of distraction rate on callus and IAN in a rabbit model of mandibular distraction osteogenesisObjective: To establish a rabbit model of simultaneous bilateral distraction of mandible. To histologically characterize new bone and inferior alveolar nerve (IAN) under different rates of distraction. Methods: 18 rabbits underwent bilateral mandibular osteotomy and placement of an internal fixator, and were divided into 0.5, 0.75 and 1.0 mm/12h groups and a sham operation group. A simplified sensory nerve test was used for evaluation of IAN function. At consolidation time of 28 d, IAN histology was studied using toluidine blue and Bodian staining and transmission electron microscopy examination. Callus specimens were subjected to radiography, von Kossa and HE staining for bone histological analysis. Results: Bone and IAN histology was closely related to the rates of distraction. Although there was no direct IAN injury caused by the surgery, each rate of distraction caused stress-based injury to IAN. 0.5 mm/12h led to the least IAN injury and the best bone formation. Conclusion: This animal model is reliable for future intervention experiments. 0.5 mm/12h may be an optimal distraction rate for simulating clinical situations.4 Histology profile of callus under slow rate of distraction in the rabbit model of mandibular DOObjective: To determine histology profile in different consolidation time under distraction rate of 0.5 mm/12h. Methods: 12 rabbits underwent gradual distraction of 0.5 mm/12h. After sequential intravital fluorochrome labelling, rabbits were sacrificed at consolidation time of 0, 14, 28 and 42 d. Bone histology was studied using fluorescence microscope, scanning electron microscopy, von Kossa staining, HE staining, and PCNA immunostaining. Results: Bone histology in this model, which began from cell aggregation of mesenchyma and ended at initial formation of mature bone, was time dependent. Conclusion: In this model, bone histology under slow rate of distraction is accord with regular pattern in DO, thus proving its reliability.5 Effects of locally injected hNGFβto new bone and IAN histology in the rabbit model of mandibular DOObjective: To evaluate the effects of local injections of hNGFβto new bone and IAN histology simultaneously in the rabbit model of mandibular DO. Methods: 20 rabbits underwent bilateral distraction at rate of 0.5 mm/12h. On the 1st and 3rd days of consolidation, 40μg hNGFβin buffer was injected into callus percutaneously for twice. The contralateral side received injections of placebo. Rabbits were sacrificed at consolidation time of either 14 or 28 d. After lateral X-rays, dimension measurements and a three-point bending test, regenerate bone was subjected to histomorphometric analysis including bone volume/total volume (BV/TV), number of osteoblasts, mineralized bone percentage and mineral apposition rate. IAN sections were stained with toluidine blue for evaluation of fiber density and mean myelinated fiber diameter and also studied using TEM. Results: hNGFβsignificantly increased maximum load, fiber density, BV/TV, mineral apposition rate of the 1st-11th day, mineralized bone percentage and fiber density compared to the control side, while number of osteoblasts and mean myelin thickness were unchanged. Conclusion: Locally applied hNGFβcan accelerate callus maturation and IAN injury recovery in the rabbit model of mandibular DO. It may therefore play a role in shortening the consolidation period and repairing/protecting IAN in mandibular DO clinically. 6 Effects of Col/nHA gel/hNGFβto new bone and IAN histology in mandibular DOObjective: To evaluate the effects of hNGFβdelivery via a Col/ nHA/k-carrageenan gel to new bone and IAN histology in the rabbit model of mandibular DO. Methods: 16 rabbits underwent bilateral distraction at rates of 0.5 or 0.75 mm/12h. On the 1st day of consolidation, the Col/ nHA/k-carrageenan gel with or without hNGFβ(20μg) was injected into callus percutaneously. The contralateral side received injections of placebo. At consolidation time of 14 d, regenerate bone specimens were subjected to BV/TV analysis and IAN specimens to fiber density and mean myelinated fiber diameter analysis. Results: The gel/ hNGFβcomposite significantly increased BV/TV at distraction rates of 0.5 and 0.75 mm/12h and fiber density at 0.5 mm/12h. New bone and IAN histology remained unchanged when treated by gel without hNGFβ. Conclusion: Local application of this gel/hNGFβcomposite can accelerate callus maturation and IAN injury recovery in the rabbit model of mandibular DO, thus reducing the consumption and injections of hNGFβ.

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