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ABSTRACT: Photodynamic therapy (PDT) has been shown to ablate tumors within vertebral bone and yield short-term improvements in vertebral architecture and biomechanical strength, in particular when combined with bisphosphonate (BP) treatment. Longer-term outcomes of PDT combined with current treatments for skeletal metastases are essential to understand its therapeutic potential. The objective of this study is to evaluate the response of vertebrae to PDT after a longer (6-week) time period, alone and combined with previous BP or radiation treatment (RT). Sixty-three female rnu/rnu rats were randomized to six treatment groups: untreated control, BP-only, RT-only, PDT-only, combined BP + PDT and combined RT + PDT. L2 vertebrae were structurally analyzed through µCT-based analysis, axial compressive load-to-failure testing and histological analysis of morphology, osteoid formation and osteoclast activity. Combined BP + PDT treatment yielded the largest improvements in bone architecture with combined RT + PDT treatment yielding similar findings, but of a lesser magnitude. Mechanically, ultimate force and stress were correlated to stereological parameters that demonstrated a positive structural effect from combinatory treatment. Increased osteoid formation was observed in both combination therapies without any significant differences in osteoclast activity. Overall, multimodality treatment demonstrated a sustained positive effect on vertebral structural integrity, motivating PDT as a minimally-invasive adjuvant treatment for spinal metastases. © 2012 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res XX:XXX-XXX, 2013.
Journal of Orthopaedic Research 04/2013; · 2.81 Impact Factor
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ABSTRACT: The second-order nonlinear polarization properties of fibrillar collagen in various rat tissues (vertebrae, tibia, tail tendon, dermis, and cornea) are investigated with polarization-dependent second-harmonic generation (P-SHG) microscopy. Three parameters are extracted: the second-order susceptibility ratio, R = [Formula: see text] ; a measure of the fibril distribution asymmetry, |A|; and the weighted-average fibril orientation, 〈δ〉. A hierarchical organizational model of fibrillar collagen is developed to interpret the second-harmonic generation polarization properties. Highlights of the model include: collagen type (e.g., type-I, type-II), fibril internal structure (e.g., straight, constant-tilt), and fibril architecture (e.g., parallel fibers, intertwined, lamellae). Quantifiable differences in internal structure and architecture of the fibrils are observed. Occurrence histograms of R and |A| distinguished parallel from nonparallel fibril distributions. Parallel distributions possessed low parameter values and variability, whereas nonparallel distributions displayed an increase in values and variability. From the P-SHG parameters of vertebrae tissue, a three-dimensional reconstruction of lamellae of intervertebral disk is presented.
Biophysical Journal 11/2012; 103(10):2093-105. · 3.65 Impact Factor
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ABSTRACT: This study compares the ability of μCT image-based registration, 2D structural rigidity analyses and multimodal continuum-level finite element (FE) modeling in evaluating the mechanical stability of healthy, osteolytic, and mixed osteolytic/osteoblastic metastatically involved rat vertebrae. μMR and μCT images (loaded and unloaded) were acquired of lumbar spinal motion segments from 15rnu/rnu rats (five per group). Strains were calculated based on image registration of the loaded and unloaded μCT images and via analysis of FE models created from the μCT and μMR data. Predicted yield load was also calculated through 2D structural rigidity analysis of the axial unloaded μCT slices. Measures from the three techniques were compared to experimental yield loads. The ability of these methods to predict experimental yield loads were evaluated and image registration and FE calculated strains were directly compared. Quantitatively for all samples, only limited weak correlations were found between the image-based measures and experimental yield load. In comparison to the experimental yield load, we observed a trend toward a weak negative correlation with median strain calculated using the image-based strain measurement algorithm (r=-0.405, p=0.067), weak significant correlations (p<0.05) with FE based median and 10th percentile strain values (r=-0.454, -0.637, respectively), and a trend toward a weak significant correlation with FE based mean strain (r=-0.366, p=0.09). Individual group analyses, however, yielded more and stronger correlations with experimental results. Considering the image-based strain measurement algorithm we observed moderate significant correlations with experimental yield load (p<0.05) in the osteolytic group for mean and median strain values (r=-0.840, -0.832, respectively), and in the healthy group for median strain values (r=-0.809). Considering the rigidity-based predicted yield load, we observed a strong significant correlation with the experimental yield load in the mixed osteolytic/osteoblastic group (r=0.946) and trend toward a moderate correlation with the experimental yield load in the osteolytic group (r=0.788). Qualitatively, strain patterns in the vertebral bodies generated using image registration and FEA were well matched, yet quantitatively a significant correlation was found only between mean strains in the healthy group (r=0.934). Large structural differences in metastatic vertebrae and the complexity of motion segment loading may have led to varied modes of failure. Improvements in load characterization, material properties assignments and resolution are necessary to yield a more generalized ability for image-based registration, structural rigidity and FE methods to accurately represent stability in healthy and pathologic scenarios.
Journal of biomechanics 08/2012; 45(14):2342-8. · 2.66 Impact Factor
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ABSTRACT: Spinal metastasis commonly occurs in advanced breast cancer. Treatment is often multimodal including radiation therapy (RT), bisphosphonates (BPs), and surgery, yet alternative minimally invasive local treatments are needed. Photodynamic therapy (PDT) has been shown to ablate tumor cells and enhance bone formation secondary to metastatic breast cancer, demonstrating potential as a treatment for spinal metastasis. Combined with previous BP treatment, bone formation was further enhanced by PDT. This study aimed to determine the effects of PDT in combination with previous RT on healthy and metastatically involved vertebrae. Forty-six athymic rats underwent RT (4 Gy on day-7), twenty-three of them were inoculated with MT-1 human breast cancer cells on day 0. Thirteen healthy and ten metastatically involved rats underwent PDT treatment on day 14. All rats were sacrificed on day 21. L2 vertebrae were analyzed using μCT imaging, mechanical testing, and histological methods. In healthy vertebrae, while modest increases in trabecular structure were found in RT + PDT compared to RT only, mechanical stability was negatively affected. The 4 Gy RT dose was found to ablate all tumor cells and prevent further vertebral metastasis. As such, in metastatically involved rats, no differences in stereological or mechanical properties were detected. RT + PDT and RT-only treatment resulted in greatly improved vertebral structural and mechanical properties versus untreated or PDT-only treatment in metastatically involved rats, due to early tumor destruction in RT-treated groups. Increased amounts of woven bone and osteoid volume were found in PDT-treated vertebrae. Further investigation is needed to understand if structural improvements seen in RT + PDT treatment can translate into longer-term improvements in strength to support the potential of PDT as a viable adjuvant treatment for spinal metastasis postradiation.
Breast Cancer Research and Treatment 07/2012; 135(2):391-401. · 4.43 Impact Factor
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ABSTRACT: STUDY DESIGN.: An in vitro biomechanical and imaging study generated from an in vivo porcine model of early stage degenerative disc disease was used to evaluate mechanical property restoration, comparing 2 minimally invasive injection techniques. OBJECTIVE.: To evaluate the ability of an injectable hydrogel to restore the mechanical properties of spinal motion segments with early stage disc degeneration, comparing 2 minimally invasive injection techniques. SUMMARY OF BACKGROUND DATA.: Treatment of early-stage disc degeneration may benefit from a combination of tissue engineering and minimally invasive therapeutic approaches. A recently developed hydrogel, thiol-modified hyaluronan elastin-like polypeptide (TMHA/EP) composite, has demonstrated potential as an injectable nucleus replacement. METHODS.: From a total of thirteen 35-kg Yorkshire boars, early-stage lumbar disc degeneration was introduced into 10 pigs via injection of chondroitinase ABC. After degeneration, 8 pigs received TMHA/EP augmentation; 1 disc via direct needle injection and a second using a modified kyphoplasty approach. High-resolution magnetic resonance images were acquired of the excised spinal motion segments, followed by biomechanical testing in axial compression, flexion-extension, lateral bending, and torsion. RESULTS.: The degenerate control motion segments were generally less stiff and more flexible than healthy controls. The injection of TMHA/EP into the degenerated nucleus produced similar mechanical stiffness to healthy controls. The direct-injected discs showed a dispersive pattern of TMHA/EP within the nucleus, whereas the modified kyphoplasty method yielded a bolus of hydrogel. Yet, mechanical behavior was comparable considering the 2 minimally invasive augmentation techniques. CONCLUSION.: The TMHA/EP composite can restore initial mechanical behavior in early-stage disc degeneration. Although both augmentation methods yielded mechanical properties comparable with healthy controls, direct injection represents a simpler technique, uses a smaller-gauge needle, does not introduce air into the disc, and yields a dispersive pattern that may be beneficial for future delivery of cells or growth factors.
Spine 07/2012; 37(20):E1296-303. · 2.08 Impact Factor
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ABSTRACT: Herein, we present a study of the pharmacokinetics and biodistribution of a butadiyne-linked conjugated porphyrin dimer (Oxdime) designed to have high near-infrared (NIR) 2-photon absorption cross-section for photodynamic therapy (PDT). Changes in biodistribution over time were monitored in mice carrying B16-F10 melanoma xenografts, following intravenous injection. Using fluorescence imaging of live animals and analyzing isolated organs ex vivo at different time points between 30 min and 24 h after injection, accumulation of Oxdime was measured in several organs (heart, kidney and liver) and in tumor. The concentration in the plasma was about 5-10 times higher than in other tissues. The fluorescence signal peaked at 3-12 h after injection in most tissues, including the tumor and the plasma. The change in the fluorescence emission spectrum of the sensitizer over time was also monitored and a shift in the maximum from 800 to 740 nm was observed over 24 h, showing that the Oxdime is metabolized. Significant quantities accumulated in the tumor, indicating that this PDT sensitizer may be promising for cancer treatment.
Photochemistry and Photobiology 06/2012; · 2.41 Impact Factor
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ABSTRACT: The vertebral column is the most frequent site of metastatic involvement of the skeleton with up to 1/3 of all cancer patients developing spinal metastases. Longer survival times for patients, particularly secondary to breast cancer, have increased the need for better understanding the impact of skeletal metastases on structural stability. This study aims to apply image registration to calculate strain distributions in metastatically involved rodent vertebrae utilizing µCT imaging. Osteolytic vertebral lesions were developed in five rnu/rnu rats 2-3 weeks post intracardiac injection with MT-1 human breast cancer cells. An image registration algorithm was used to calculate and compare strain fields due to axial compressive loading in metastatically involved and control vertebrae. Tumor-bearing vertebrae had greatly increased compressive strains, double the magnitude of strain compared to control vertebrae (p=0.01). Qualitatively strain concentrated within the growth plates in both tumor bearing and control vertebrae. Most interesting was the presence of strain concentrations at the dorsal wall in metastatically involved vertebrae, suggesting structural instability. Strain distributions, quantified by image registration were consistent with known consequences of lytic involvement. Metastatically involved vertebrae had greater strain magnitude than control vertebrae. Strain concentrations at the dorsal wall in only the metastatic vertebrae, were consistent with higher incidence of burst fracture secondary to this pathology. Future use of image registration of whole vertebrae will allow focused examination of the efficacy of targeted and systemic treatments in reducing strains and the related risk of fracture in pathologic bones under simple and complex loading.
Journal of Orthopaedic Research 12/2011; 30(7):1032-9. · 2.81 Impact Factor
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ABSTRACT: Spinal metastases often show mixed areas of enhanced (osteoblastic) bone growth adjacent to areas of thinning (osteolytic) bone. This study aims to quantitatively characterize bone quality and tumor burden within a new rat model of mixed osteolytic/osteoblastic spinal metastases. Mixed vertebral metastases were analyzed in nude rats 21-days post intracardiac injection of Ace-1 canine prostate cancer cells. Vertebral micro-architecture was assessed in µCT images. Histologic processing quantified tumor burden (PTHrP), osteoclast activity (TRAP), and osteoid formation (Goldner's Trichrome) in ½ of all samples. Remaining samples were mechanically tested to failure in compression. Metastatically involved vertebrae exhibited extreme osteolysis, evident through an increase in osteoclasts leading to significantly reduced trabecular bone volume. Metastatically involved vertebrae also exhibited increased osteoid characteristic of osteoblastic lesions. While mechanical properties in tumor-bearing vertebrae were not significantly decreased compared to controls, a strong correlation was found between trabecular volumetric BMD and ultimate force. The highly aggressive Ace-1 skeletal metastases demonstrated predominant osteolysis with some areas of immature, new osteoblastic bone formation. Bone quality resulting from these lesions consisted of decreased structural properties, but without a significant impact on mechanical integrity.
Journal of Orthopaedic Research 10/2011; 30(5):817-23. · 2.81 Impact Factor
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ABSTRACT: Skeletal metastases most frequently affect the vertebral column and may lead to severe consequences including fracture. Clinical management of skeletal metastases often utilizes a multimodal treatment approach, including bisphosphonates (BPs). Previous work has demonstrated the synergistic potential of photodynamic therapy (PDT) in combination with BP in treating osteolytic disease through structural, histologic, and destructive mechanical testing analyses. Recent work has developed and validated image-based methods that may be used to non-destructively determine mechanical stability in whole bones, and enable their use for additional (i.e. histologic) analysis. In this work we use an intensity-based 3D image registration technique to compare the strain patterns throughout untreated control and BP + PDT treated rnu/rnu rat spinal motion segments with osteolytic metastases. It was hypothesized that the combination treatment will reduce average and maximum strain values and restore the pattern of strain to that of healthy vertebrae. Mean, median, and 90th percentile strains in the control group were significantly higher than the treatment group. High strain areas in both groups were observed around the endplates; in the control group, large areas of high strains were also observed around the lesions and adjacent to the dorsal wall. Absence of high strains adjacent to the dorsal wall (similar to healthy vertebrae) may correspond to a reduced risk of burst fracture following BP + PDT therapy. This study demonstrates the application of non-destructive image analysis to quantify the positive mechanical effects of combined BP + PDT treatment in the metastatic spine.
Annals of biomedical engineering 08/2011; 39(11):2816-22. · 2.41 Impact Factor
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ABSTRACT: Breast cancer is the second leading cause of cancer-related death in women. Approximately 85% of patients with advanced cases will develop spinal metastases. The vertebral column is the most common site of breast cancer metastases, where overexpression of matrix metalloproteinases (MMPs) promotes the spread of cancer. Current therapies have significant limitations due to the high associated risk of damaging the spinal cord. An attractive alternative is photodynamic therapy providing noninvasive and site-selective treatment. However, current photosensitizers are limited by their nonspecific accumulation. Photodynamic molecular beacons (PP(MMP)B), activated by MMPs, offer another level of PDT selectivity and image-guidance preserving criticial tissues, specifically the spinal cord. Metastatic human breast carcinoma cells, MT-1, were used to model the metastatic behavior of spinal lesions. In vitro and in vivo evidence demonstrates MMP specific activation of PP(MMP)B in MT-1 cells. Using a clinically relevant metastatic model, fluorescent imaging establishes the specific activation of PP(MMP)B by vertebral metastases versus normal tissue (i.e., spinal cord) demonstrating the specificity of these beacons. Here, we validate that the metastasis-selective mechanism of PP(MMP)Bs can specifically image breast cancer vertebral metastases, thereby differentiating tumor and healthy tissue.
Bioconjugate Chemistry 06/2011; 22(6):1021-30. · 4.93 Impact Factor
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ABSTRACT: Breast cancer patients commonly develop metastases in the spine, which compromises its mechanical stability and can lead to skeletal related events. The current clinical standard of treatment includes the administration of systemic bisphosphonates (BP) to reduce metastatically induced bone destruction. However, response to BPs can vary both within and between patients, which motivates the need for additional treatment options for spinal metastasis. Photodynamic therapy (PDT) has been shown to be effective at treating metastatic lesions secondary to breast cancer in an athymic rat model, and is proposed as a treatment for spinal metastasis. The objective of this study was to determine the effect of PDT, alone or in combination with previously administered systemic BPs, on the structural and mechanical integrity of both healthy and metastatically involved vertebrae. Human breast carcinoma cells (MT-1) were inoculated into athymic rats (day 0). At 14 days, a single PDT treatment was administered, with and without previous BP treatment at day 7. In addition to causing tumor necrosis in metastatically involved vertebrae, PDT significantly reduced bone loss, resulting in strengthening of the vertebrae compared to untreated controls. Combined treatment with BP + PDT further enhanced bone architecture and strength in both metastatically involved and healthy bone. Overall, the ability of PDT to both ablate malignant tissue and improve the structural integrity of vertebral bone motivates its consideration as a local minimally invasive treatment for spinal metastasis secondary to breast cancer.
Breast Cancer Research and Treatment 11/2010; 124(1):111-9. · 4.43 Impact Factor
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ABSTRACT: In autofluorescence endoscopy, the difference in the fluorescence of intrinsic fluorophores is imaged to help visualize pre-malignant lesions, as in the system evaluated here. In this, blue light is used for excitation and the green autofluorescence is normalized by the red diffuse reflectance and presented using a false color scale. The present study was designed to quantify the degree of fluorescence photobleaching induced by the excitation light during use in the colon, since significant photobleaching could lead to false interpretation of the images, particularly false-positive lesions.
Measurements were made ex vivo and in vivo, both using the endoscopic imaging system and a separate fiberoptic spectroscopy probe in externalized rat jejunum and in patients undergoing routine colonoscopy, using exposures typical of autofluorescence endoscopic examination.
Photobleaching could be potentially caused at blue light exposure. However, at light intensities and exposure times that are typically used in clinical practice, the average photobleaching (% loss of peak fluorescence intensity) was <1% and <6% in the rat and human tissues, respectively. Nevertheless, the range was large: from -17% to +18% in rats and -33% to +43% in patients, where negative values denote an apparent increase in fluorescence. Both the large positive and negative deviations are believed in part to be due to a measurement artifact caused by uncontrollable tissue motility.
It is concluded that, using exposures typically encountered in clinical practice, there is minimal photobleaching during fluorescence endoscopy at exposure such as are used in the Onco-LIFE and comparable systems. The small changes in fluorescence intensity and spectral shift that do occur are not likely to be detectable by eye, and so should not impact significantly on the diagnostic accuracy of the technique.
Lasers in Surgery and Medicine 03/2010; 42(3):224-31. · 2.75 Impact Factor
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ABSTRACT: This study investigates the effects of photodynamic therapy (PDT) on the structural integrity of vertebral bone in healthy rats.
To determine the short-term (1 week) and intermediate term (6 weeks) effects of a single PDT treatment on the mechanical and structural properties of vertebral bone.
Spinal metastasis develops in up to one-third of all cancer patients, compromising the mechanical integrity of the spine and thereby increasing the risk of pathologic fractures and spinal cord damage. PDT has recently been adapted to ablate metastatic tumors in the spine in preclinical animal models. However, little is known about the effects of PDT on the structural integrity of vertebral bone.
A single PDT treatment was administered to healthy Wistar rats at photosensitizer and light doses known to be effective in athymic rats bearing human breast cancer metastases. At both 1 and 6 weeks posttreatment, changes in trabecular architecture, global stiffness and strength of vertebrae were quantified using micro-CT stereological analysis and axial compression testing.
At 6 weeks, there was a significant increase in bone volume fraction (to 55.7 +/- 11.1% vs. 38.5 +/- 6.4%, P < 0.001) and decrease in bone surface area-to-volume ratio (16.9 +/- 5.0/mm vs. 22.8 +/- 4.5/mm, P = 0.001), attributed to trabecular thickening (130 +/- 40 microm vs. 90 +/- 20 microm, P < 0.001). Similar trends were found at 1 week after PDT. There was a significant increase in stiffness from control (306 +/- 123 N/mm) at 1 week (399 +/- 150 N/mm, P = 0.04) and 6 weeks (410 +/- 113 N/mm, P = 0.05) post PDT. There was a positive trend toward increased ultimate stress at 1 week, which became statistically significant at 6 weeks compared with control (39.3 +/- 11.3 MPa vs. 27.5 +/- 9.5 MPa control, P = 0.002).
Not only may PDT be successful in ablating metastatic tumor tissue in the spine, but the positive effects of PDT on bone found in this study suggest that PDT may also improve vertebral mechanical stability.
Spine 02/2010; 35(3):272-7. · 2.08 Impact Factor
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ABSTRACT: To assess the antimicrobial elution characteristics, toxicity, and antimicrobial activity of amikacin-impregnated ferric-hyaluronate implants (AI-FeHAI) for amikacin delivery to the tarsocrural joint of horses.
Experimental study.
AI-FeHAI implants, equine cartilage, and synovium, and horses (n=6).
In vitro study: Five AI-FeHAI were placed in saline solution with daily replacement until implant degradation. Eluent was tested for amikacin concentration and bioactivity. Synovial and cartilage explants were incubated in the presence or absence of AI-FeHAI for 72 hours and subsequently assessed for morphology, viability, and composition. Synovial explants were incubated with Staphylococcus aureus in the presence or absence of AI-FeHAI. Spent medium was cultured daily and explants were assessed for morphology and viability after 96 hours. In vivo study: AI-FeHAI were placed in 6 tarsocrural joints. Standard cytologic analysis and amikacin concentration (SFAC) were determined in synovia obtained regularly for 28 days thereafter. Similar analyses were conducted after a single intra-articular injection of amikacin 6 months later.
In vitro study: Amikacin concentrations exceeded 16 microg/mL and inhibited S. aureus growth for 8 days. AI-FeHAI had no effect on cartilage explants. AI-FeHAI eliminated bacteria from synovial explants. In vitro study: After AI-FeHAI placement, SFAC was highest (140.78+63.81 microg/mL) at first sampling time. By 24 hours SFAC was <16 microg/mL. After intra-articular injection, SFAC was the highest (377.91 +/- 40.15 microg/mL) at first sampling time. By 48 hours SFAC was <16 microg/mL.
A single intra-articular amikacin injection demonstrated superior pharmacokinetics than AI-FeHAI prepared as described.
AI-FeHAI cannot be recommended for clinical use.
Veterinary Surgery 06/2009; 38(4):498-505. · 1.26 Impact Factor
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ABSTRACT: Bovine articular cartilage is often used to study chondrocytes in vitro. It is difficult to correlate in vitro studies using bovine chondrocytes with in vivo studies using other species such as rabbits and sheep. The aim of this investigation was to study the effect of species, anatomical location and exogenous growth factors on chondrocyte proliferation in vitro.
Equine (EQ), bovine (BO) and ovine (OV) articular chondrocytes from metacarpophalangeal (fetlock (F)), shoulder (S) and knee (K) joints were cultured in tissue culture flasks. Growth factors (rh-FGFb: 10 ng/ml; rh-TGFbeta: 5 ng/ml) were added to the cultures at days 2 and 4. On day 6, cells were counted and flow cytometry analysis was performed to determine cell size and granularity. A three factor ANOVA with paired Tukey's correction was used for statistical analysis.
After 6 days in culture, cell numbers had increased in control groups of EQ-F, OV-S, OV-F and BO-F chondrocytes. The addition of rh-FGFb led to the highest increase in cell numbers in the BO-F, followed by EQ-F and OV-S chondrocytes. The addition of rh-TGFbeta increased cell numbers in EQ-S and EQ-F chondrocytes, but showed nearly no effect on EQ-K, OV-K, OV-S, OV-F and BO-F chondrocytes. There was an overall difference with the addition of growth factors between the different species and joints.
Different proliferation profiles of chondrocytes from the various joints were found. Therefore, we recommend performing in vitro studies using the species and site where subsequent in vivo studies are planned.
BMC Musculoskeletal Disorders 02/2005; 6:23. · 1.58 Impact Factor
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ABSTRACT: Photodynamic therapy has been successfully applied to numerous cancers. Its potential to treat cancer metastases in the spine has been demonstrated previously in a preclinical animal model. The aim of this study was to test two photosensitizers, benzoporphyrin-derivative monoacid ring A (BPD-MA) and by 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), for their potential use to treat bony metastases. The difference in photosensitizer concentration in the spinal cord and the surrounding tumor-bearing vertebrae was of particular interest to assess the risk of potential collateral damage to the spinal cord. Vertebral metastases in a rat model were generated by intracardiac injection of human breast cancer cells. When tumor growth was confirmed, photosensitizers were injected systemically and the animals were euthanized at different time points. The following tissues were harvested: liver, kidney, ovaries, appendicular bone, spinal cord and lumbar vertebrae. Photosensitizer tissue concentration of BPD-MA or PpIX was determined by fluorescence spectrophotometry. In contrast to BPD-MA, ALA-PpIX did not demonstrate an appreciable difference in the uptake ratio in tumor-bearing vertebrae compared to spinal cord. The highest ratio for BPD-MA concentration was found 15 min after injection, which can be recommended for therapy in this model.
Photochemistry and Photobiology 83(5):1034-9. · 2.41 Impact Factor
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ABSTRACT: This study was designed to demonstrate that bioluminescence imaging (BLI) can be used as a new tool to evaluate the effects of low-level laser therapy (LLLT) during in vivo inflammatory process. Here, the efficacy of LLLT in modulating inducible nitric oxide synthase (iNOS) expression using different therapeutic wavelengths was determined using transgenic animals with the luciferase gene under control of the iNOS gene expression. Thirty transgenic mice, FVB/N-Tg(iNOS-luc)Xen, were allocated randomly to one of four experimental groups treated with different wavelengths (lambda = 635, 785, 808 and 905 nm) or a control group (nontreated). Inflammation was induced by intra-articular injection of zymosan A in both knee joints. Laser treatment (25 mW cm(-2), 200 s, 5 J cm(-2)) was applied to the knees 15 min after inflammation induction. Measurements of iNOS expression were performed at various times (0, 3, 5, 7, 9 and 24 h) by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The results showed a significant increase in BLI signal after irradiation with 635 nm laser when compared to the nonirradiated animals and the other LLLT-treated groups, indicating wavelength dependence of LLLT effects on iNOS expression during the inflammatory process, and thus demonstrating an action spectrum of iNOS gene expression following LLLT in vivo that can be detected by BLI. Histological analysis was also performed and demonstrated the presence of fewer inflammatory cells in the synovial joints of mice irradiated with 635 nm compared with nonirradiated knee joints.
Photochemistry and Photobiology 81(6):1351-5. · 2.41 Impact Factor
