Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep

BACKGROUND: Articular cartilage has limited capacity to repair. Defects greater than 3 mm heal with formation of inferior fibrous cartilage. Therefore, many attempts have been made to find the ideal graft for larger cartilage lesions. Different grafts, such as untreated or cryopreserved osteochondral transplants, have been used with variable success. METHODS: Photo-oxidized osteochondral grafts were implanted in both femoral condyles of one ovine knee. Untreated xenogeneic and autogeneic grafts served as controls. Three groups of 8 sheep each were formed and they were sacrificed 6, 12 or 18 months after surgery. RESULTS: The macroscopic evaluation of the condyle and graft showed a well-maintained cartilage surface in most grafts at all time points. However, the host cartilage matrix deteriorated considerably in all xenogeneic, most autogeneic and fewer of the photo-oxidized grafts at 12 and 18 months, respectively. The blue colour of the photo-oxidized grafts resulting from the process of photo-oxidation was visible in all grafts at 6 months, had diminished at 12 months and had completely disappeared at 18 months after surgery. Histologically a loss of matrix staining was almost never noticed in untreated xenografts, transiently at 6 months in photo-oxidized grafts and increased at 12 and 18 months. Fusion between graft and host cartilage could be seen in photo-oxidized grafts at 12 and 18 months, but was never seen in autografts and xenografts. CONCLUSIONS: The photo-oxidation of osteochondral grafts and its use as transplant appears to have a beneficial effect on cartilage and bone remodelling. Osteochondral grafts pre-treated with photo-oxidation may be considered for articular cartilage replacement and therefore may delay artificial joint replacements in human patients

μFE models can represent microdamaged regions of healthy and metastatically involved whole vertebrae identified through histology and contrast enhanced μCT imaging

Micro-damage formation within the skeleton is an important stimulant for bone remodeling, however abnormal build-up of micro-damage can lead to skeletal fragility. In this study, µCT imaging based micro finite element (μFE) models were used to evaluate tissue level damage criteria in whole healthy and metastatically-involved vertebrae. T13-L2 spinal segments were excised from osteolytic (n=3) and healthy (n=3) female athymic rnu/rnu rats. Osteolytic metastasis was generated by intercardiac injection of HeLa cancer cells. Micro-mechanical axial loading was applied to the spinal motion segments under μCT imaging. Vertebral samples underwent BaSO4 staining and sequential calcein/fuchsin staining to identify load induced micro-damage. μCT imaging was used generate specimen specific μFE models of the healthy and osteolytic whole rat vertebrae. Model boundary conditions were generated through deformable image registration of loaded and unloaded scans. Elevated stresses and strains were detected in regions of micro-damage identified through histological and BaSO4 staining within healthy and osteolytic vertebral models, as compared to undamaged regions. Additionally, damaged regions of metastatic vertebrae experienced significantly higher local stresses and strains than those in the damaged regions of healthy specimens. Areas identified by BaSO4 staining, however, yielded lower levels of stress and strain in damaged and undamaged regions of healthy and metastatic vertebrae as compared to fuschin staining. The multimodal (experimental, image-based and computational) techniques used in this study demonstrated the ability of local stresses and strains computed through µFE analysis to identify trabecular micro-damage, that can be applied to biomechanical analyses of healthy and diseased whole bones

Quantitative MRI assessment of VX2 tumour oxygenation changes in response to hyperoxia and hypercapnia

Magnetic resonance imaging (MRI) relaxation times provide indirect estimates of tissue O(2) for monitoring tumour oxygenation. This study provides insight into mechanisms underlying longitudinal (R(1) = 1/T(1)) and transverse effective (R(2)* = 1/T(2)*) relaxation rate changes during inhalation of 100% O(2) and 3%, 6% and 9% CO(2) (balanced O(2)) in a rabbit tumour model. Quantitative R(1), R(2)*, and dynamic contrast-enhanced (DCE) imaging was performed in six rabbits 12-23 days following implantation of VX2 carcinoma cells in the quadricep muscle. Invasive measurements of tissue partial pressure of O(2) (pO(2)) and perfusion were also performed, which revealed elevated pO(2) levels in all tumour regions for all hyperoxic gases compared to baseline (air) and reduced perfusion for carbogen. During 100% O(2) breathing, an R(1) increase and R(2)* decrease consistent with elevated pO(2) were observed within tumours. DCE-derived blood flow was weakly correlated with R(1) changes from air to 100% O(2). Further addition of CO(2) (carbogen) did not introduce considerable changes in MR relaxation rates, but a trend towards higher R(1) relative to breathing 100% O(2) was observed, while R(2)* changes were inconsistent. This observation supports the predominance of dissolved O(2) on R(1) sensitivity and demonstrates the value of R(1) over R(2)* for tissue oxygenation measures.The work is supported by the grant from the Sponsors Canadian Institutes of Health Research

Can micro-imaging based analysis methods quantify structural integrity of rat vertebrae with and without metastatic involvement?

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

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

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

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

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

Abstract 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