The purpose of the study was to examine the effects of vascular-targeted photodynamic therapy (PDT) using benzoporhyrin derivative (BPD) on growth plates in spine and long bones. Specifically we wish to determine whether the ipsilateral up-regulation of VEGF in the thoracic and/or lumbar spine following treatment with leads to onset of scoliosis morphologically similar to idiopathic adolescent scoliosis. And secondly confirm growth plate closure in long bones following BPD-PDT resulting in leg length discrepancy.

A 0.2 mm fiber was placed through an 18g needle onto one side of the distal femoral epiphysis (n=24) or lower thoracic/upper lumbar vertebral bodies of four-week old mice (n=18). Mice are genetically modified to emit bioluminescence upon activation of the vascular endothelial growth factor gene (VEGF). Accurate placement was confirmed using fluoroscopy. BPD (2 mg/Kg, i.v.) was administered systemically and the growth plates were stimulated with 690nm laser light five minutes later. Range of light dose regimens were tested. Animals were followed for a total of seven-twelve weeks post treatment. Faxitron imaging and bioluminescent imaging were obtained to determine leg length or curve progression and VEGF activity. Histology and immunohistochemistry including H& E, HIF-1á, CD31 and VEGF immunohistochemistry was performed.

PDT was able to up-regulate VEGF for up to four weeks following treatment following a percutaneous treatment using a 0.2mm treatment fiber both in the femur and vertebrae. Femoral shortening occurred with histological evidence of bone formation across the growth plate. We were able to identify using faxitron abnormal curvature in a number of the animals that received 5J, 10 mW regimen.

This study confirms that that the epiphyses of vertebrae and long bones are similarly susceptible to the effects of a hypoxic insult resulting in VEGF up-regulation. We are proposing that this stress response can lead to premature closure of epiphyseal growth plates of long bones resulting in limb growth arrest or asymmetric growth of vertebrae and the development of scoliosis in an animal model.

Purpose: This study investigates the use of photodynamic therapy (PDT) in regulating bone development with a view to its potential role in treating Juvenile leg length discrepancy (LLD).

Methods: Transgenic mice expressing the luciferase firefly gene upon activation of a promoter sequence specific to the vascular endothelial growth factor (VEGF) gene were subject to benzoporphyrin derivative monoacid (BPD-MA)-mediated PDT in the right, tibial epiphyseal growth plate at the age of 3 weeks. BPD-MA was administered intracardially (2mg/kg) followed 10 mins later by a laser light (690 +/− 5 nm) at a range of doses (5-27J, 50 mW output) delivered either as a single or repeat regimen (x2-3). Contra-lateral legs served as no-light controls. Further controls included animals that received light treatment in the absence of photosensitizer or no treatment. Mice were imaged for VEGF related bioluminescence (photons/sec/steradian) at t= 0, 24, 48, 72 h and 1–4 weeks post PDT. FaxitronÔ x-ray images provided accurate assessment of bone morphometry. Upon sacrifice, the tibia and femur of the treated and untreated limbs were harvested, imaged and measured again and prepared for histology. A number of animals were sacrificed at 24 h post PDT to allow immunohistochemical staining for CD31, VEGF and hypoxia-inducible factor (HIF-1 alpha) within the bone.

Results: PDT-treated (10 J, x2) mice displayed enhanced bioluminescence at the treatment site (and ear nick) for up to 4 weeks post treatment while control mice were bioluminescent at the ear-nick site only. Repeat regimens provided greater shortening of the limb than the corresponding single treatment. PDT-treated limbs were shorter by 3–4 mm on average as compared to the contra lateral and light only controls (10 J, x2). Immunohistochemistry confirmed the enhanced expression VEGF and CD31 at 4 weeks post-treatment although no increase in HIF-1& #945; was evident at either 24 h or 4 weeks post PDT treatment.

Conclusions: Results confirm the utility of PDT to provide localized effects on bone development that may be applicable to other related skeletal deformities.