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.

Treatment affects load to failure and microdamage accumulation in healthy and osteolytic rat vertebrae.

Bone turnover and microdamage are impacted by the presence of skeletal metastases which can contribute to increased fracture risk. Treatments for metastatic disease may further impact bone quality. This exploratory study aimed to establish an initial understanding of microdamage accumulation and load to failure in healthy and osteolytic rat vertebrae following focal and systemic cancer treatment (docetaxel (DTX), stereotactic body radiotherapy (SBRT), or zoledronic acid (ZA)). Osteolytic spine metastases were developed in 6-week-old athymic female rats via intracardiac injection of HeLa human cervical cancer cells (day 0). Additional rats served as healthy controls. Rats were either untreated, received SBRT to the T10-L6 vertebrae on day 14 (15 Gy, two fractions), DTX on day 7 or 14, or ZA on day 7. Rats were euthanized on day 21. Tumor burden was assessed with bioluminescence images acquired on day 14 and 21, histology of the excised T11 and L5 vertebrae, and ex-vivo µCT images of the T13-L4. Microstructural parameters (bone volume/total volume, trabecular number, spacing, thickness, and bone mineral density) were measured from L2 vertebrae. Load to failure was measured with axial compressive loading of the L1-L3 motion segments. Microdamage accumulation was labeled in T13 vertebrae with BaSO4 staining and was visualized with high resolution µCT imaging. Microdamage volume fraction was defined as the ratio of BaSO4 to bone volume. DTX administered on day 7 reduced tumor growth significantly (p < 0.05). Microdamage accumulation was found to be increased by the presence of metastases but was reduced by all treatments with ZA showing the largest improvement in HeLa cell injected rats. Load to failure was decreased in untreated and SBRT HeLa cell injected rats compared to healthy controls (p < 0.01). There was a moderate negative correlation between load to failure and microdamage volume fraction in vertebrae from rats injected with HeLa cells (R = -0.35, p = 0.031). Strong correlations were also found between microstructural parameters and load to failure and microdamage accumulation. Several factors, including the presence of osteolytic lesions and use of cancer therapies, influence microdamage accumulation and load to failure in rat vertebrae. Understanding the impact of these treatments on fracture risk of metastatic vertebrae is important to improve management of patients with spinal metastases.

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.

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.

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.

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.

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.

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.

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.

Photodynamic Therapy for the Treatment of Vertebral Metastases: A Phase I Clinical Trial.

PURPOSE: Vertebroplasty (VP) and balloon kyphoplasty (KP) are minimally invasive stabilization procedures for pathologic vertebral compression fractures (VCF). Concurrent administration of photodynamic therapy (PDT) as a tumor-ablative modality has yet to be studied in humans as a potential complement to improved mechanical stability that is afforded by vertebral cement augmentation (VCA). PATIENTS AND METHODS: This first-in-human trial used a single 6 mg/m2 dose of the clinical photosensitizer Visudyne with escalating laser light doses. Following a cohort of light-only controls (n = 6), the drug and light treatment groups (n = 6 each) were 50, 100, 150, and 200 J/cm. VCA was performed within 15 minutes following PDT. Patients were clinically reviewed at 1 and 6 weeks. The primary outcome measure was safety from a neurologic perspective. RESULTS: Thirty patients comprising a variety of primary tumors were treated with PDT and either KP or VP. Vertebral PDT was technically feasible and delivered in all study patients. No dose groups showed significant increases in pain as defined by the generic SF-36 as well as disease-specific EORTC-QLQ-BM22 and EORTC-QLQ-C15-PAL questionnaires. The 50 and 100 J/cm groups showed the most significant pain reduction (P < 0.05). Twelve (40%) patients experienced complications during the study including 3 patients with further vertebral fracture progression by 6 weeks despite VCA. No complications were directly attributed to PDT. CONCLUSIONS: Using the parameters described, vertebral PDT as an adjunct to VCA is safe from a pharmaceutical and neurologic perspective. The results of this trial motivate scale-up study evaluating potential PDT efficacy in vertebral metastatic treatment.

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.

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.

Mechanical behavior of metastatic vertebrae are influenced by tissue architecture, mineral content, and organic feature alterations.

Diminished vertebral mechanical behavior with metastatic involvement is typically attributed to modified architecture and trabecular bone content. Previous work has identified organic and mineral phase bone quality changes in the presence of metastases, yet limited work exists on the potential influence of such tissue level modifications on vertebral mechanical characteristics. This work seeks to determine correlations between features of bone (structural and tissue level) and mechanical behavior in metastatically involved vertebral bone. It is hypothesized that tissue level properties (mineral and organic) will improve these correlations beyond architectural properties and BMD alone. Twenty-four female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic (N = 8) or mixed (osteolytic/osteoblastic, N = 7) metastases, respectively. Twenty-one days post-inoculation L1-L3 pathologic vertebral motion segments were excised and µCT imaged. 3D morphometric parameters and axial rigidity of the L2 vertebrae were quantified. Sequential loading and µCT imaging measured progression of failure, stiffness and peak force. Relationships between mechanical testing (whole bone and tissue-level) and tissue-level material property modifications with metastatic involvement were evaluated utilizing linear regression models. Osteolytic involvement reduced vertebral trabecular bone volume, structure, CT-derived axial rigidity, stiffness and failure force compared to healthy controls (N = 9). Mixed metastases demonstrated similar trends. Previously assessed collagen cross-linking and proline-based residues were correlated to mechanical behavior and improved the predictive ability of the regression models. Similarly, collagen organization improved predictive regression models for metastatic bone hardness. This work highlights the importance of both bone content/architecture and organic tissue-level features in characterizing metastatic vertebral mechanics. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3013-3022, 2018.

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.

Collagen fibril organization within rat vertebral bone modified with metastatic involvement.

Metastatic involvement diminishes the mechanical integrity of vertebral bone, however its specific impact on the structural characteristics of a primary constituent of bone tissue, the collagen-I fibril matrix, has not been adequately characterized. Female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic or mixed (osteolytic & osteoblastic) metastases respectively. A maximum of 21days was allowed between inoculation and rat sacrifice for vertebrae extraction. Linear polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and transmission electron microscopy (TEM) imaging was utilized to assess the impact of metastatic involvement on collagen fibril organization. Increased observations of deviations in the typical plywood motif or a parallel packing structure and an increased average measured susceptibility ratio (related to relative degree of in-plane vs. out-plane fibrils in the analyzed tissue area) in bone adjacent to metastatic involvement was indicative of change in fibrilar organization compared to healthy controls. In particular, collagen-I fibrils in tumour-induced osteoblastic bone growth showed no adherence to the plywood motif or parallel packing structure seen in healthy lamellar bone, exhibiting a much higher susceptibility ratio and degree of fibril disorder. Negative correlations were established between measured susceptibility ratios and the hardness and modulus of metastatic bone tissue assessed in a previous study. Characterizing modifications in tissue level properties is key in defining bone quality in the presence of metastatic disease and their potential impact on material behaviour.

The impact of metastasis on the mineral phase of vertebral bone tissue.

The negative impact of metastases on the mechanical performance of vertebral bone is often attributed to reduced bone density and/or compromised architecture. However limited characterization has been done on the impact of metastasis on the mineralization of bone tissue and resulting changes in material behaviour. This study aimed to evaluate the impact of metastasis on micro and nano scale characteristics of the mineral phase of bone, specifically mineral crystal growth, homogeneity of mineralization and changes in intrinsic material properties. Female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic or mixed (osteolytic & osteoblastic) metastases respectively (N=17 per group). A maximum of 21 days was allowed between inoculation and sacrifice of inoculated rats and healthy age-matched uninoculated controls (N=11). X-ray diffraction was used to assess average crystal size in crushed L1-L3 vertebrae; backscatter electron microscopy and nanoindentation were utilized to evaluate modifications in bone mineral density distribution and material behaviour (tissue hardness and modulus) in sagittal-sectioned, embedded and polished L5 vertebrae. HeLa inoculated samples showed reduced mineral crystal width compared to healthy controls. While both types of metastatic involvement reduced tissue mineral content, pathological osteoblastic bone, specific to Ace-1 inoculated samples, significantly decreased tissue mineral homogeneity, whereas osteolytic bone from HeLa samples saw a slight increase in homogeneity. The modulus and hardness of pathological osteoblastic bone was diminished compared to all other bone. Elucidating changes in material behaviour and mineralization of bone tissue is key to defining bone quality in the presence of metastatic involvement.

Post-euthanasia micro-computed tomography-based strain analysis is able to represent quasi-static in vivo behavior of whole vertebrae.

Three-dimensional image-based strain measurement in whole bones allows representation of physiological, albeit quasi-static, loading conditions. However, such work to date has been limited to specimens postmortem. The main purpose of this study is to verify the efficacy of deformable image registration of post-euthanasia strain to characterize the in vivo mechanical behavior of rat vertebrae. A micro-computed tomography-compatible custom loading device was used to apply 75 N load to a three-level caudal motion segment of a healthy rat. Loaded and unloaded micro-computed tomography scans were acquired in vivo and post-sacrifice. A micro-computed tomography-based deformable image registration algorithm was used to calculate vertebral strains live and post-euthanasia. No significant difference was found in the in vivo strains (-0.011 ± 0.001) and ex vivo strains (-0.012 ± 0.001) obtained from the comparisons of loaded and unloaded images (p = 0.3). Comparisons between unloaded-unloaded and loaded-loaded scans yielded significantly lower axial strains, representing the error of the method. Qualitatively, high strains were observed adjacent to growth plate regions in evaluating the loaded-unloaded images. Strain patterns in the loaded-loaded and unloaded-unloaded scans were inconsistent as would be expected in representing noise. Overall, live and dead loaded to unloaded comparisons yielded similar strain patterns and magnitudes. Point-wise differences in axial strain fields also supported this observation. This study demonstrated a proof of concept, suggesting that post-euthanasia micro-computed tomography-based strain analysis is able to represent the in vivo quasi-static behavior of rat tail vertebrae.