|Title:||Effect of low intensity pulsed ultrasound on rhBMP-4 induced osteogenesis in a spinal fusion model|
|Subject:||Hong Kong Polytechnic University -- Dissertations.|
Ultrasonics in medicine.
Bones -- Growth.
|Department:||Department of Rehabilitation Sciences|
|Pages:||xx, 174 leaves : ill. (some col.) ; 30 cm.|
|Abstract:||Low intensity pulsed ultrasound (LIPU) has been proven effective in enhancing bone repair. It is well known that LIPU can effectively enhance bone healing in fresh fracture, delayed-union or non-union, bone defect and bone distraction. However, few studies have reported the effect of LIPU on ectopic ossification, which is the formation of bone in extraskeletal tissues. Moreover, the precise biological mechanisms underlying the effect of LIPU enhanced bone repair remain unclear. LIPU is a form of acoustic wave that can be transmitted through and into biological tissue to deliver mechanical energy. It is important therefore, to relate the mechanical basis of ultrasound to the sensitivity of the target biological tissues to the mechanical stimuli through its mechano-sensory nerve fibers which secrete calcitonin gene related peptide (CGRP). To date, little evidence is available on this relationship. The aim of the present study is to investigate the effect of LIPU on the recombinant human bone morphogenetic protein-4 (rhBMP-4) induced ectopic ossification and the role of CGRP-positive nerve in ectopic ossification and its response to ultrasound stimulation. Sixty 30-week old (around 4.5kg) New Zealand white rabbits were used as the experimental animals for this study. All animals underwent a single level non-decorticated bilateral posterolateral intertransverse process fusion at L5-L6 by implantation of poly (d,l-lactic acid) (PDLLA) incorporated with rhBMP-4. LIPU treatment was applied unilaterally on the body surface over the implantation site daily, starting at day 2 after operation. Rabbits were randomly allocated into 10 groups according to the application of different doses of rhBMP-4 (low dose or high dose) and different treatment durations of LIPU (3 days, 1 week, 3 weeks, 7 weeks and 12 weeks). Animals were sacrificed after the LIPU treatment was completed. The new bone formation at the fusion site was assessed using radiography, peripheral quantitative computed tomography (pQCT) and microscopy after animals to be sacrificed at the end of the experiment. The innervation of CGRP expressing sensory nerve in the fusion area was detected through immunofluorescent microscopy. The x-ray, pQCT and light microscopy results showed that LIPU treatment could significantly enhance rhBMP-4 induced ectopic ossification in the present study. New bone formation was observed via intramembranous ossification and endochondral ossification on the sham LIPU side of all groups, indicating that the application of rhBMP-4 could effectively induce ectopic ossification. Moreover, more new bone tissues were found in the high dose rhBMP-4 groups than in low dose rhBMP-4 groups at week 7 and week 12 postoperation, suggesting the apparent dose-dependent effect of rhBMP-4 on inducing and promoting ectopic ossification. On the other hand, LIPU treatment effectively enhanced rhBMP-4 induced ectopic ossification. The pQCT results showed that bone mineral content (BMC) and bone volume were significantly higher on the LIPU treated side than those on the sham LIPU side at week 7 and week 12 postoperation in both the low and high dose rhBMP-4 groups. Histological results also showed that the percentage of new cartilage tissue was significantly higher on the LIPU treated side than that on the sham LIPU side at week 3 and 7 postoperation, suggesting that LIPU could enhance rhBMP-4 induced ectopic ossification by improving endochondral ossification. Fluorescent microcopy results showed that LIPU treatment enhanced the innervation of CGRP-positive nerve fibers in ectopic bone tissues. Spatially, LIPU promoted the in-growth of CGRP-positive nerve fibers into the new bone forming tissues. On the sham LIPU side, CGRP-positive nerve fibers were observed only at the fibrous tissues which surrounded the new bone and cartilage tissues, and in the bone marrow of the new bone tissue. On the LIPU treated side, CGRP-positive nerve fibers were also observed in the new bone and cartilage tissues in addition to the presence in fibrous and bone marrow tissues. Temporally, LIPU increased the density of CGRP-positive nerve fibers in fibrous and bone marrow tissues during the process of ectopic ossification. On the sham LIPU side, the density of CGRP-positive nerve fibers in fibrous tissues was of a low level at day 3 and week 1, and then increased markedly at week 3. On the LIPU treated side, LIPU treatment increased the density of CGRP-positive nerve fibers to a high level from day 3, suggesting that the promotion effect of LIPU on the in-growth of CGRP-positive nerve fibers into the ectopic bone was more effectively during the early stage of healing. The present study revealed that LIPU could effectively enhance rhBMP-4 induced ectopic ossification, possibly by improving endochondral ossification. Moreover, LIPU treatment facilitated the invasion of CGRP-positive sensory fibers in ectopic ossification during the healing process, which contributes to the overall concept of mechanobiology and its application in augmenting repair of musculoskeletal tissues in conjunction with its associated regulatory system(s).|
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