Polarimetric second harmonic generation microscopy of partially oriented fibers II: Imaging study.

Polarimetric second harmonic generation (SHG) microscopy imaging is employed to investigate the ultrastructural organization of biological and biomimetic partially oriented fibrillar structures. The linear polarization-in polarization-out SHG microscopy measurements are conducted with rat tail tendon, rabbit cornea, pig cartilage, and biomimetic meso-tetra(4-sulfonatophenyl)porphine (TPPS4) cylindrical aggregates, which represent different two- and three-dimensional (2D and 3D) configurations of C6 symmetry fibril structures in the focal volume (voxel) of the microscope. The polarization-in polarization-out imaging of rat tail tendon reveals that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and achiral (R) and chiral (C) susceptibility component ratio values change by tilting the tendon fibers out of image plane. The R ratio changes for the 2D crossing fibers observed in cornea tissue. The 3D crossing of fibers also affects R ratio in cartilage tissue. The distinctly different dependence of R on crossing and tilting of fibers is demonstrated in collagen and TPPS4 aggregates, due to the achiral molecular susceptibility ratio having values below and above 3, respectively. The polarimetric microscopy results correspond well with the analytical expressions of amplitude and R and C ratios dependence on the crossing angle of the fibers. The experimentally measured SHG intensity and R and C ratio maps are consistent with the computational modeling of various fiber configurations presented in the preceding article. The demonstrated SHG intensity and R and C ratio dependencies on fibril configurations provide the basis for interpreting polarimetric SHG microscopy images in terms of 3D ultrastructural organization of fibers in each voxel of the samples.

The impact of thermal cycling on Staphylococcus aureus biofilm growth on stainless steel and titanium orthopaedic plates.

BACKGROUND: Orthopaedic implant infections are difficult to eradicate because bacteria adhering to implant surfaces inhibit the ability of the immune system and antibiotics to combat these infections. Thermal cycling is a temperature modulation process that improves performance and longevity of materials through molecular structural reorientation, thereby increasing surface uniformity. Thermal cycling may change material surface properties that reduce the ability for bacteria to adhere to the surface of orthopaedic implants. This study aims to determine whether thermal cycling of orthopaedic implants can reduce bacterial growth. METHODS: In a randomized, blinded in-vitro study, titanium and stainless steel plates treated with thermal cycling were compared to controls. Twenty-seven treated and twenty-seven untreated plates were covered with 10 ml tryptic soy broth containing ~ 105 colony forming units (CFU)/ml of bioluminescent Staphylococcus aureus (S. aureus)Xen29 and incubated at 37 °C for 14d. Quantity and viability of bacteria were characterized using bioluminescence imaging, live/dead staining and determination of CFUs. RESULTS: Significantly fewer CFUs grow on treated stainless steel plates compared to controls (p = 0.0088). Similar findings were seen in titanium plates (p = 0.0048) following removal of an outlier. No differences were evident in live/dead staining using confocal microscopy, or in metabolic activity determined using bioluminescence imaging (stainless steel plates: p = 0.70; titanium plates: p = 0.26). CONCLUSION: This study shows a reduction in CFUs formation on thermal cycled plates in-vitro. Further in-vivo studies are necessary to investigate the influence of thermal cycling on bacterial adhesion during bone healing. Thermal cycling has demonstrated improved wear and strength, with reductions in fatigue and load to failure. The added ability to reduce bacterial adhesions demonstrates another potential benefit of thermal cycling in orthopaedics, representing an opportunity to reduce complications following fracture fixation or arthroplasty.

In vitroassessment of a gallium-doped glass polyalkenoate cement: chemotherapeutic potential, cytotoxicity and osteogenic effects.

Metastatic bone lesions are often osteolytic, which causes advanced-stage cancer sufferers to experience severe pain and an increased risk of developing a pathological fracture. Gallium (Ga) ion possesses antineoplastic and anti-bone resorption properties, suggesting the potential for its local administration to impede the growth of metastatic bone lesions. This study investigated the chemotherapeutic potential, cytotoxicity, and osteogenic effects of a Ga-doped glass polyalkenoate cement (GPC) (C-TA2) compared to its non-gallium (C-TA0) counterpart. Ion release profiles revealed a biphasic pattern characterized by an initial burst followed by a gradually declining release of ions. C-TA2 continued to release Ga steadily throughout the experimentation period (7 d) and exhibited prolonged zinc (Zn) release compared to C-TA0. Interestingly, the Zn release from both GPCs appeared to cause a chemotherapeutic effect against H1092 lung cancer cellsin vitro, with the prolonged Zn release from C-TA2 extending this effect. Unfortunately, both GPCs enhanced the viability of HCC2218 breast cancer cells, suggesting that the chemotherapeutic effects of Zn could be tied to cellular differences in preferred Zn concentrations. The utilization of SAOS-2 and MC3T3 cell lines as bone cell models yielded conflicting results, with the substantial decline in MC3T3 viability closely associated with silicon (Si) release, indicating cellular variations in Si toxicity. Despite this ambiguity, both GPCs exhibited harmful effects on the osteogenesis of primary rat osteoblasts, raising concerns about excessive burst Zn release. While Ga/Zn-doped GPCs hold promise for treating metastatic bone lesions caused by lung cancers, further optimization is required to mitigate cytotoxicity on healthy bone.

Development and validation of a radiofrequency ablation treatment planning system for vertebral metastases.

PURPOSE: Bone-targeted radiofrequency ablation (RFA) is widely used in the treatment of vertebral metastases. While radiation therapy utilizes established treatment planning systems (TPS) based on multimodal imaging to optimize treatment volumes, current RFA of vertebral metastases has been limited to qualitative image-based assessment of tumour location to direct probe selection and access. This study aimed to design, develop and evaluate a computational patient-specific RFA TPS for vertebral metastases. METHODS: A TPS was developed on the open-source 3D slicer platform, including procedural setup, dose calculation (based on finite element modelling), and analysis/visualization modules. Usability testing was carried out by 7 clinicians involved in the treatment of vertebral metastases on retrospective clinical imaging data using a simplified dose calculation engine. In vivo evaluation was performed in a preclinical porcine model (n = 6 vertebrae). RESULTS: Dose analysis was successfully performed, with generation and display of thermal dose volumes, thermal damage, dose volume histograms and isodose contours. Usability testing showed an overall positive response to the TPS as beneficial to safe and effective RFA. The in vivo porcine study showed good agreement between the manually segmented thermally damaged volumes vs. the damage volumes identified from the TPS (Dice Similarity Coefficient = 0.71 ± 0.03, Hausdorff distance = 1.2 ± 0.1 mm). CONCLUSION: A TPS specifically dedicated to RFA in the bony spine could help account for tissue heterogeneities in both thermal and electrical properties. A TPS would enable visualization of damage volumes in 2D and 3D, assisting clinicians in decisions about potential safety and effectiveness prior to performing RFA in the metastatic spine.

Wide-field Stokes polarimetric microscopy for second harmonic generation imaging.

We employ wide-field second harmonic generation (SHG) microscopy together with nonlinear Stokes polarimetry for quick ultrastructural investigation of large sample areas (700 µm × 700 µm) in thin histology sections. The Stokes vector components for SHG are obtained from the polarimetric measurements with incident and outgoing linear and circular polarization states. The Stokes components are used to construct the images of polarimetric parameters and deduce the maps of ultrastructural parameters of achiral and chiral nonlinear susceptibility tensor components ratios and cylindrical axis orientation in fibrillar materials. The large area imaging was employed for lung tumor margin investigations. The imaging shows reduced SHG intensity, increased achiral susceptibility ratio values, and preferential orientation of collagen strands along the boarder of tumor margin. The wide-field Stokes polarimetric SHG microscopy opens a possibility of quick large area imaging of ultrastructural parameters of tissue collagen, which can be used for nonlinear histopathology investigations.

Hierarchical model of fibrillar collagen organization for interpreting the second-order susceptibility tensors in biological tissue.

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.

Beyond radiation therapy: photodynamic therapy maintains structural integrity of irradiated healthy and metastatically involved vertebrae in a pre-clinical in vivo model.

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.

Establishment and Image based evaluation of a New Preclinical Rat Model of Osteoblastic Bone Metastases.

Bone remodeling is disrupted in the presence of metastases and can present as osteolytic, osteoblastic or a mixture of the two. Established rat models of osteolytic and mixed metastases have been identified changes in structural and tissue-level properties of bone. The aim of this work was to establish a preclinical rat model of osteoblastic metastases and characterize bone quality changes through image-based evaluation. Female athymic rats (n = 22) were inoculated with human breast cancer cells ZR-75-1 and tumor development tracked over 3-4 months with bioluminescence and in-vivo µCT imaging. Bone tissue-level stereological features were quantified on ex-vivo µCT imaging. Histopathology verified the presence of osteoblastic bone. Bone mineral density distribution was assessed via backscattered electron microscopy. Newly formed osteoblastic bone was associated with reduced mineral content and increased heterogeneity leading to an overall degraded bone quality. Characterizing changes in osteoblastic bone properties is relevant to pre-clinical therapeutic testing and treatment planning.

Irregular porous titanium enhances implant stability and bone ingrowth in an intra-articular ovine model.

Two commercially available porous coatings, Gription and Porocoat, were compared for the first time in a challenging intra-articular, weight-bearing, ovine model. Gription has evolved from Porocoat and has higher porosity, coefficient of friction, and microtextured topography, which are expected to enhance bone ingrowth. Cylindrical implants were press-fit into the weight-bearing regions of ovine femoral condyles and bone ingrowth and fixation strength evaluated 4, 8, and 16 weeks postoperatively. Biomechanical push-out tests were performed on lateral femoral condyles (LFCs) to evaluate the strength of the bone-implant interface. Bone ingrowth was assessed in medial femoral condyles (MFCs) as well as implants retrieved from LFCs following biomechanical testing using backscattered electron microscopy and histology. By 16 weeks, Gription-coated implants exhibited higher force (2455 ± 1362 vs. 1002 ± 1466 N; p = 0.046) and stress (12.60 ± 6.99 vs. 5.14 ± 7.53 MPa; p = 0.046) at failure, and trended towards higher stiffness (11,510 ± 7645 vs. 5010 ± 8374 N/mm; p = 0.061) and modulus of elasticity (591 ± 392 vs. 256 ± 431 MPa; p = 0.061). A strong, positive correlation was detected between bone ingrowth in LFC implants and failure force (r = 0.93, p < 10-13 ). By 16 weeks, bone ingrowth in Gription-coated implants in MFCs was 10.50 ± 6.31% compared to 5.88 ± 2.77% in Porocoat (p = 0.095). Observations of the bone-implant interface, made following push-out testing, showed more bony material consistently adhered to Gription compared to Porocoat at all three time points. Gription provided superior fixation strength and bone ingrowth by 16 weeks.

Imaging of specific activation of photodynamic molecular beacons in breast cancer vertebral metastases.

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.

Impact of radiofrequency ablation (RFA) on bone quality in a murine model of bone metastases.

Thermal therapies such as radiofrequency ablation (RFA) are gaining widespread clinical adoption in the local treatment of skeletal metastases. RFA has been shown to successfully destroy tumor cells, yet the impact of RFA on the quality of the surrounding bone has not been well characterized. RFA treatment was performed on femora of rats with bone metastases (osteolytic and osteoblastic) and healthy age matched rats. Histopathology, second harmonic generation imaging and backscatter electron imaging were used to characterize changes in the structure, organic and mineral components of the bone after RFA. RFA treatment was shown to be effective in targeting tumor cells and promoting subsequent new bone formation without impacting the surrounding bone negatively. Mineralization profiles of metastatic models were significantly improved post-RFA treatment with respect to mineral content and homogeneity, suggesting a positive impact of RFA treatment on the quality of cancer involved bone. Evaluating the impact of RFA on bone quality is important in directing the growth of this minimally invasive therapeutic approach with respect to fracture risk assessment, patient selection, and multimodal treatment planning.

Photodynamic therapy outcome modelling for patients with spinal metastases: a simulation-based study.

Spinal metastases often occur in the advanced stages of breast, lung or prostate cancer, resulting in a significant impact on the patient's quality of life. Current treatment modalities for spinal metastases include both systemic and localized treatments that aim to decrease pain, improve mobility and structural stability, and control tumour growth. With the development of non-toxic photosensitizer drugs, photodynamic therapy (PDT) has shown promise as a minimally invasive non-thermal alternative in oncology, including for spinal metastases. To apply PDT to spinal metastases, predictive algorithms that optimize tumour treatment and minimize the risk of spinal cord damage are needed to assess the feasibility of the treatment and encourage a broad acceptance of PDT in clinical trials. This work presents a framework for PDT modelling and planning, and simulates the feasibility of using a BPD-MA mediated PDT to treat bone metastases at two different wavelengths (690 nm and 565 nm). An open-source software for PDT planning, PDT-SPACE, is used to evaluate different configurations of light diffusers (cut-end and cylindrical) fibres with optimized power allocation in order to minimize the damage to spinal cord or maximize tumour destruction. The work is simulated on three CT images of metastatically involved vertebrae acquired from three patients with spinal metastases secondary to colorectal or lung cancer. Simulation results show that PDT at a 565 nm wavelength has the ability to treat 90% of the metastatic lesion with less than 17% damage to the spinal cord. However, the energy required, and hence treatment time, to achieve this outcome with the 565 nm is infeasible. The energy required and treatment time for the longer wavelength of 690 nm is feasible ([Formula: see text] min), but treatment aimed at 90% of the metastatic lesion would severely damage the proximal spinal cord. PDT-SPACE provides a simulation platform that can be used to optimize PDT delivery in the metastatic spine. While this work serves as a prospective methodology to analyze the feasibility of PDT for tumour ablation in the spine, preclinical studies in an animal model are ongoing to elucidate the spinal cord damage extent as a function of PDT dose, and the resulting short and long term functional impairments. These will be required before there can be any consideration of clinical trials.

Beyond bisphosphonates: photodynamic therapy structurally augments metastatically involved vertebrae and destroys tumor tissue.

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.

Defining the therapeutic window of vertebral photodynamic therapy in a murine pre-clinical model of breast cancer metastasis using the photosensitizer BPD-MA (Verteporfin).

Breast cancer is known to cause metastatic lesions in the bone, which can lead to skeletal-related events. Currently, radiation therapy and surgery are the treatment of choice, but the success rate varies and additional adjuncts are desirable. Photodynamic therapy (PDT) has been applied successfully as a non-radiative treatment for numerous cancers. Earlier work has shown that the athymic rat model is suitable to investigate the effect of PDT on bone metastasis and benzoporphyrin-derivative monoacid ring A (BPD-MA; verteporfin) has been shown to be a selective photosensitizer. The aim of this study was to define the therapeutic window of photosensitizer with regard to drug and light dose. Human breast carcinoma cells (MT-1)-stable transfected with the luciferase gene-were injected intra-cardiacally into athymic rats. At 14 days, the largest vertebral lesion by bioluminescence imaging was targeted for single treatment PDT. A drug escalating-de-escalating scheme was used (starting drug dose and light energy of 0.2 mg/kg and 50 J, respectively). Outcomes included 48 h post-treatment bioluminescence of remaining viable tumour, histomorphometric assessment of tumour burden, and neurologic evaluation. The region of effect by bioluminescence and histology increased with increasing drug dose and light energy. A safe and effective drug-light dose combination in this model appears to be 0.5 mg/kg BPD-MA and applied light energy of less than 50 J for the thoracic spine and 1.0 mg/kg and 75 J for the lumbar spine. For translation to clinical use, it is an advantage that BPD-MA (verteporfin), a second-generation photosensitizer, is already approved to treat age-related macular degeneration. Overall, PDT represents an exciting potential new minimally-invasive local, safe and effective therapy in the management of patients with spinal metastases.

Assessment of photobleaching during endoscopic autofluorescence imaging of the lower GI tract.

BACKGROUND AND OBJECTIVES: 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. STUDY DESIGN: 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. RESULTS: 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. SUMMARY AND CONCLUSIONS: 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.

In vitro and in vivo evaluation of ferric-hyaluronate implants for delivery of amikacin sulfate to the tarsocrural joint of horses.

OBJECTIVE: 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. STUDY DESIGN: Experimental study. SAMPLE POPULATION: AI-FeHAI implants, equine cartilage, and synovium, and horses (n=6). METHODS: 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. RESULTS: 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. CONCLUSIONS: A single intra-articular amikacin injection demonstrated superior pharmacokinetics than AI-FeHAI prepared as described. CLINICAL RELEVANCE: AI-FeHAI cannot be recommended for clinical use.

Elevated Microdamage Spatially Correlates with Stress in Metastatic Vertebrae.

Metastasis of cancer to the spine impacts bone quality. This study aims to characterize vertebral microdamage secondary to metastatic disease considering the pattern of damage and its relationship to stress and strain under load. Osteolytic and mixed osteolytic/osteoblastic vertebral metastases were produced in athymic rats via HeLa cervical or canine Ace-1 prostate cancer cell inoculation, respectively. After 21 days, excised motion segments (T12-L2) were µCT scanned, stained with BaSO4 and re-imaged. T13-L2 motion segments were loaded in axial compression to induce microdamage, re-stained and re-imaged. L1 (loaded) and T12 (unloaded) vertebrae were fixed, sample blocks cut, polished and BSE imaged. µFE models were generated of all L1 vertebrae with displacement boundary conditions applied based on the loaded µCT images. µCT stereological analysis, BSE analysis and µFE derived von Mises stress and principal strains were quantitatively compared (ANOVA), spatial correlations determined and patterns of microdamage assessed qualitatively. BaSO4 identified microdamage was found to be spatially correlated with regions of high stress in µFEA. Load-induced microdamage was shown to be elevated in the presence of osteolytic and mixed metastatic disease, with diffuse, crossed hatched areas of microdamage present in addition to linear microdamage and microfractures in metastatic tissue, suggesting diminished bone quality.

Complex Susceptibilities and Chiroptical Effects of Collagen Measured with Polarimetric Second-Harmonic Generation Microscopy.

Nonlinear optical properties of collagen type-I are investigated in thin tissue sections of pig tendon as a research model using a complete polarimetric second-harmonic generation (P-SHG) microscopy technique called double Stokes-Mueller polarimetry (DSMP). Three complex-valued molecular susceptibility tensor component ratios are extracted. A significant retardance is observed between the chiral susceptibility component and the achiral components, while the achiral components appear to be in phase with each other. The DSMP formalism and microscopy measurements are further used to explain and experimentally validate the conditions required for SHG circular dichroism (SHG-CD) of collagen to occur. The SHG-CD can be observed with the microscope when: (i) the chiral second-order susceptibility tensor component has a non-zero value, (ii) a phase retardance is present between the chiral and achiral components of the second-order susceptibility tensor and (iii) the collagen fibres are tilted out of the image plane. Both positive and negative areas of SHG-CD are observed in microscopy images, which relates to the anti-parallel arrangement of collagen fibres in different fascicles of the tendon. The theoretical formalism and experimental validation of DSMP imaging technique opens new opportunities for ultrastructural characterisation of chiral molecules, in particular collagen, and provides basis for the interpretation of SHG-CD signals. The nonlinear imaging of chiroptical parameters offers new possibilities to further improve the diagnostic sensitivity and/or specificity of nonlinear label-free histopathology.

Collagen chirality and three-dimensional orientation studied with polarimetric second-harmonic generation microscopy.

Polarization-dependent second-harmonic generation (P-SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three-dimensional (3D) orientation map of collagen fibers within a pig tendon. C6 symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference χzzz2/χzxx2 and χxyz2/χzxx2 , where the latter is a newly extracted parameter from the P-SHG images and is related to the chiral structure of collagen. The χxyz2/χzxx2 is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P-SHG imaging was performed using a linear polarization-in polarization-out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation-independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells.

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.