Bone turnover and microdamage are impacted by skeletal metastases which can contribute to increased fracture risk. Treatments for metastatic disease may further impact bone quality. This study aimed to establish an understanding of microdamage accumulation and load to failure in healthy and osteolytic vertebrae following cancer treatment (stereotactic body radiotherapy (SBRT), zoledronic acid (ZA), or docetaxel (DTX)).

Forty-two 6-week old athymic female rats (Hsd:RH-Foxn1rnu, Envigo) were studied; 22 were inoculated with HeLa cervical cancer cells through intracardiac injection (day 0). Animals were randomly assigned to four groups: untreated (healthy=5, osteolytic=6), SBRT on day 14 (healthy=6, osteolytic=6), ZA on day 7 (healthy=4, osteolytic=5), and DTX on day 14 (healthy=5, osteolytic=5). Animals were euthanized on day 21. L1-L3 motion segments were compression loaded to failure and force-displacement data recorded. T13 vertebrae were stained with BaSO₄ and µCT imaged (90kVp, 44uA, 4.9µm) to visualize microdamage location and volume. Damage volume fraction (DV/BV) was calculated as the ratio of BaSO₄ to bone volume. Differences in mean load-to-failure were compared using three-way ANOVA (disease status, treatment, cells injected). Differences in mean DV/BV between treatment groups were compared using one-way ANOVA.

Treatment had a significant effect on load-to-failure (p=0.004) with ZA strengthening the healthy and osteolytic vertebrae. Reduced strength post SBRT seen in the metastatic (but not the healthy) group may be explained by greater tumor involvement secondary to higher cell injection concentrations. Untreated metastatic samples had higher DV/BV (16.25±2.54%) compared to all treatment groups (p<0.05) suggesting a benefit of treatment to bone quality.

Focal and systemic cancer treatments were shown to effect load-to-failure and microdamage accumulation in healthy and osteolytic vertebrae. Developing a better understanding of how treatments effect bone quality and mechanical stability is critical for effective management of patients with spinal metastases.

Bone turnover and the accumulation of 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. The present study aims to establish a preliminary understanding of microdamage accumulation and load to failure in osteolytic vertebrae following stereotactic body radiotherapy (SBRT), zoledronic acid (ZA), or docetaxel (DTX) treatment.

Twenty-two six-week old athymic female rats (Hsd:RH-Foxn1rnu, Envigo, USA) were inoculated with HeLa cervical cancer cells through intracardiac injection (day 0). Institutional approval was obtained for this work and the ARRIVE guidelines were followed. Animals were randomly assigned to four groups: untreated (n=6), spine stereotactic body radiotherapy (SBRT) administered on day 14 (n=6), zoledronic acid (ZA) administered on day 7 (n=5), and docetaxel (DTX) administered on day 14 (n=5). Animals were euthanized on day 21. T13-L3 vertebral segments were collected immediately after sacrifice and stored in −20°C wrapped in saline soaked gauze until testing. µCT scans (µCT100, Scanco, Switzerland) of the T13-L3 segment confirmed tumour burden in all T13 and L2 vertebrae prior to testing. T13 was stained with BaSO₄ to label microdamage. High resolution µCT scans were obtained (90kVp, 44uA, 4W, 4.9µm voxel size) to visualize stain location and volume. Segmentations of bone and BaSO₄ were created using intensity thresholding at 3000HU (~736mgHA/cm³) and 10000HU (~2420mgHA/cm³), respectively. Non-specific BaSO₄ was removed from the outer edge of the cortical shell by shrinking the segmentation by 105mm in 3D. Stain volume fraction was calculated as the ratio of BaSO₄ volume to the sum of BaSO₄ and bone volume. The L1-L3 motion segments were loaded under axial compression to failure using a µCT compatible loading device (Scanco) and force-displacement data was recorded. µCT scans were acquired unloaded, at 1500µm displacement and post-failure. Stereological analysis was performed on the L2 vertebrae in the unloaded µCT scans. Differences in mean stain volume fraction, mean load to failure, and mean bone volume/total volume (BV/TV) were compared between treatment groups using one-way ANOVAs. Pearson's correlation between stain volume fraction and load to failure by treatment was calculated using an adjusted load to failure divided by BV/TV.

Stained damage fraction was significantly different between treatment groups (p=0.0029). Tukey post-hoc analysis showed untreated samples to have higher stain volume fraction (16.25±2.54%) than all treatment groups (p<0.05). The ZA group had the highest mean load to failure (195.60±84.49N), followed by untreated (142.33±53.08N), DTX (126.60±48.75N), and SBRT (95.50±44.96N), but differences did not reach significance (p=0.075). BV/TV was significantly higher in the ZA group (49.28±3.56%) compared to all others. The SBRT group had significantly lower BV/TV than the untreated group (p=0.018). Load divided by BV/TV was not significantly different between groups (p=0.24), but relative load to failure results were consistent (ZA>Untreated>DTX>SBRT). No correlations were found between stain volume fraction and load to failure.

Focal and systemic cancer treatments effect microdamage accumulation and load to failure in osteolytic vertebrae. Current testing of healthy controls will help to further separate the effects of the tumour and cancer treatments on bone quality.

Bone turnover and the accumulation of 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. The present study aims to establish a preliminary understanding of microdamage accumulation and load to failure in osteolytic vertebrae following stereotactic body radiotherapy (SBRT), zoledronic acid (ZA), or docetaxel (DTX) treatment.

Twenty-two six-week old athymic female rats (Hsd:RH-Foxn1rnu, Envigo, USA) were inoculated with HeLa cervical cancer cells through intracardiac injection (day 0). Institutional approval was obtained for this work and the ARRIVE guidelines were followed. Animals were randomly assigned to four groups: untreated (n=6), spine stereotactic body radiotherapy (SBRT) administered on day 14 (n=6), zoledronic acid (ZA) administered on day 7 (n=5), and docetaxel (DTX) administered on day 14 (n=5). Animals were euthanized on day 21. T13-L3 vertebral segments were collected immediately after sacrifice and stored in −20°C wrapped in saline soaked gauze until testing. µCT scans (µCT100, Scanco, Switzerland) of the T13-L3 segment confirmed tumour burden in all T13 and L2 vertebrae prior to testing. T13 was stained with BaSO₄ to label microdamage. High resolution µCT scans were obtained (90kVp, 44uA, 4W, 4.9µm voxel size) to visualize stain location and volume. Segmentations of bone and BaSO₄ were created using intensity thresholding at 3000HU (~736mgHA/cm³) and 10000HU (~2420mgHA/cm³), respectively. Non-specific BaSO₄ was removed from the outer edge of the cortical shell by shrinking the segmentation by 105mm in 3D. Stain volume fraction was calculated as the ratio of BaSO₄ volume to the sum of BaSO₄ and bone volume. The L1-L3 motion segments were loaded under axial compression to failure using a µCT compatible loading device (Scanco) and force-displacement data was recorded. µCT scans were acquired unloaded, at 1500µm displacement and post-failure. Stereological analysis was performed on the L2 vertebrae in the unloaded µCT scans. Differences in mean stain volume fraction, mean load to failure, and mean bone volume/total volume (BV/TV) were compared between treatment groups using one-way ANOVAs. Pearson's correlation between stain volume fraction and load to failure by treatment was calculated using an adjusted load to failure divided by BV/TV.

Stained damage fraction was significantly different between treatment groups (p=0.0029). Tukey post-hoc analysis showed untreated samples to have higher stain volume fraction (16.25±2.54%) than all treatment groups (p<0.05). The ZA group had the highest mean load to failure (195.60±84.49N), followed by untreated (142.33±53.08N), DTX (126.60±48.75N), and SBRT (95.50±44.96N), but differences did not reach significance (p=0.075). BV/TV was significantly higher in the ZA group (49.28±3.56%) compared to all others. The SBRT group had significantly lower BV/TV than the untreated group (p=0.018). Load divided by BV/TV was not significantly different between groups (p=0.24), but relative load to failure results were consistent (ZA>Untreated>DTX>SBRT). No correlations were found between stain volume fraction and load to failure.

Focal and systemic cancer treatments effect microdamage accumulation and load to failure in osteolytic vertebrae. Current testing of healthy controls will help to further separate the effects of the tumour and cancer treatments on bone quality.

Patients undergoing revision surgery of a primary total hip arthroplasty often exhibit bone loss and poor bone quality, which make achieving stable fixation and osseointegration challenging. Implant components coated in porous metals are used clinically to improve mechanical stability and encourage bone in-growth. We compared ultra-porous titanium coatings, known commercially as Gription and Porocoat, in an intra-articular model by press-fitting coated cylindrical implants into ovine femoral condyles and evaluating bone in-growth and fixation strength 4, 8 and 16 weeks post-operatively.

Bilateral surgery using a mini-arthrotomy approach was performed on twenty-four Dorset-Rideau Arcott rams (3.4 ± 0.8 years old, 84.8 ± 9.3 kg) with Institutional Animal Care Committee approval in accordance with the Canadian Council on Animal Care. Cylindrical implants, 6.2 mm in diameter by 10 mm in length with surface radius of curvature of 35 mm, were composed of a titanium substrate coated in either Porocoat or Gription and press-fit into 6 mm diameter recipient holes in the weight-bearing regions of the medial (MFC) and lateral (LFC) femoral condyles. Each sheep received 4 implants; two Gription in one stifle (knee) and two Porocoat in the contralateral joint. Biomechanical push-out tests (Instron ElectroPuls E10000) were performed on LFCs, where implants were pushed out relative to the condyle at a rate of 2 mm/min. Force and displacement data were used to calculate force and displacement at failure, stiffness, energy, stress, strain, elastic modulus, and toughness. MFCs were fixed in 70% ethanol, processed undecalcified, and polished sections, approximately 70 µm thick (Exakt Micro Grinding system) were carbon-coated. Backscattered electron images were collected on a scanning electron microscope (Hitachi SU3500) at 5 kV and working distance of 5 mm. Bone in-growth within the porous coating was quantified using software (ImageJ). Statistical comparisons were made using a two-way ANOVA and Fisher's LSD post-hoc test (Statistica v.8).

Biomechanical evaluation of the bone-implant interface revealed that by 16 weeks, Gription-coated implants exhibited higher force (2455±1362 N vs. 1002±1466 N, p=0.046) and stress (12.60±6.99 MPa vs. 5.14±7.53 MPa, p=0.046) at failure, and trended towards higher stiffness (11510±7645 N/mm vs. 5010±8374 N/mm, p=.061) and modulus of elasticity (591±392 MPa vs. 256±431 MPa, p=0.61). Similarly, by 16 weeks, bone in-growth in Gription-coated implants was approximately double that measured in Porocoat (6.73±3.86 % vs. 3.22±1.52 %, p=0.045). No statistically significant differences were detected at either 4 nor 8 weeks, however, qualitative observations of the exposed bone-implant interface, made following push-out testing, showed more bony material consistently adhered to Gription compared to Porocoat at all three time points. High variability is attributed to implant placement, resulting from the small visual window afforded during surgery, unique curvatures of the condyles, and presence of the extensor digitorum longus tendon which limited access to the LFC.

Ultra-porous titanium coatings, know commercially as Gription and Porocoat, were compared for the first time in a challenging intra-articular ovine model. Gription provided superior fixation strength and bone in-growth, suggesting it may be beneficial in hip replacement surgeries where bone stock quality and quantity may be compromised.

Quantitative assessment of metastatic involvement of the bony spine is important for assessing disease progression and treatment response. Quantification of metastatic involvement is challenging as tumours may appear as osteolytic (bone resorbing), osteoblastic (bone forming) or mixed. This investigation aimed to develop an automated method to accurately segment osteoblastic lesions in a animal model of metastatically involved vertebrae, imaged with micro computed tomography (μCT).

Radiomics seeks to apply standardized features extracted from medical images for the purpose of decision-support as well as diagnosis and treatment planning. Here we investigate the application of radiomic-based features for the delineation of osteoblastic vertebral metastases. Osteoblastic lesions affect bone deposition and bone quality, resulting in a change in the texture of bony material physically seen through μCT imaging. We hypothesize that radiomics based features will be sensitive to changes in osteoblastic lesion bone texture and that these changes will be useful for automating segmentation.

Osteoblastic metastases were generated via intracardiac injection of human ZR-75-1 breast cancer cells into a preclinical athymic rat model (n=3). Four months post inoculation, ex-vivo μCT images (µCT100, Scanco) were acquired of each rodent spine focused on the metastatically involved third lumbar vertebra (L3) at 7µm/voxel and resampled to 34µm/voxel.

The trabecular bone within each vertebra was isolated using an atlas and level-set based segmentation approach previously developed by our group. Pyradiomics, an open source Radiomics library written in python, was used to calculate 3D image features at each voxel location within the vertebral bone. Thresholding of each radiomic feature map was used to isolate the osteoblastic lesions.

The utility of radiomic feature-based segmentation of osteoblastic bone tissue was evaluated on randomly selected 2D sagittal and axial slices of the μCT volume. Feature segmentations were compared to ground truth osteoblastic lesion segmentations by calculating the Dice Similarity Coefficient (DSC). Manually defined ground truth osteoblastic tumor segmentations on the μCT slices were informed by histological confirmation of the lesions.

The radiomic based features that best segmented osteoblastic tissue while optimizing computational time were derived from the Neighbouring Gray Tone Difference Matrix (NGTDM). Measures of coarseness yielded the best agreement with the manual segmentations (DSC=707%) followed by contrast, strength and complexity (DSC=6513%, 5428%, and 4826%, respectively).

This pilot study using a radiomic based approach demonstrates the utility of the NGTDM features for segmentation of vertebral osteoblastic lesions. This investigation looked at the utility of isolated features to segment osteoblastic lesions and found modest performance in isolation. In future work we will explore combining these features using machine learning based classifiers (i.e. decision forests, support vector machines, etc.) to improve segmentation performance.

Summary

A novel bipolar cooled radiofrequency ablation probe, optimised for bone metastases applications, is shown in two preclinical models to offer a safe and minimally invasive treatment option that can ablate large tissue volumes and preserve the regenerative ability of bone.

Purpose

Maintenance of vertebral mechanical stability is of paramount importance to prevent pathologic fractures and resultant neurologic compromise in individuals with spinal metastases. Current non-surgical treatments for vertebral metastases (i.e. chemotherapy, bisphophonates (BP) and radiation) yield variable responses in the tumour and surrounding bone. Photodynamic therapy (PDT) is a novel, minimally-invasive technology that utilizes a drug activated by light at a specific non-thermal wavelength to locally destroy tumour cells. Previously, we observed that PDT can ablate cancer cells within bone and yield short-term (1-week) improvements in vertebral architecture and biomechanical strength, particularly when combined with BP therapy. This study aims to evaluate the effects of PDT in vertebral bone over a longer (6-week) time period, alone and combined with previous BP treatment, to determine if improvements in skeletal architecture and strength are maintained.

Purpose

Nucleus pulposus (NP) replacements represent a less invasive alternative for treatment of early stage degenerative disc disease (DDD). Hydrogel based NP replacements are of particular interest as they can be injected/implanted using minimally invasive surgical (MIS) techniques to re-establish mechanical integrity and as a scaffold for regeneration. A thiol-modified hyaluronan elastin-like polypeptide (TMHA/EP) hydrogel crosslinked using polyethylene diacrylate has shown promise as a potential NP replacement for DDD in vitro. This study aims to assess the mechanical properties of this hydrogel when injected into an induced early stage DDD porcine model and to determine the optimal injection method for delivery. It is hypothesized that minimally invasive injection of the TMHA/EP material can restore mechanical behaviour of spinal motion segments in early stage DDD.

Rodents are often used as preclinical models for investigating the biomechanical consequences of spinal pathologies and interventions. Growth plates are present within rat vertebrae throughout life and may alter the vertebral biomechanics. This study investigates the biomechanical response of rat-tail vertebrae to axial compressive loading using μCT imaging and image registration to spatially resolve strain fields.

The sixth caudal vertebrae of eight immunocompromised (rnu/rnu) rats were μCT scanned (17.5 ×17.5×17.5μm/pixel) in both loaded (27N-32N axial compression) and unloaded configurations. Image registration was used to calculate strain and displacement fields in the bone due to the applied load by finding a spatial mapping between the two scans. Strain was resolved to varying spatial resolutions; high strain gradient regions, such as the growth plates, were analyzed to higher spatial resolutions.

Axial strains calculated by image registration ranged from 2% in tension to 16% in compression with an average axial strain of 1.6% in compression. In seven rats the majority of the strain measured within the vertebrae was concentrated in the growth plate. Very soft growth plates in three specimens resulted in maximum axial strains from 10–16% in compression. The remaining four rats with strain concentrations in the growth plate had maximum axial strains ranging from 2.2%–3.2%. Centrally located strain concentrations of lower magnitudes and more limited spatial extent were observed in the trabecular bone.

The majority of the strain within the rat vertebrae was absorbed by the growth plates. The amount of strain within the growth plate is important to consider when interpreting biomechanical data on rat vertebrae. Load application to rodent vertebrae will first compress the growth plate and only following compression of this structure cause significant development of displacement and strains within the trabecular and cortical bone. This insight into the biomechanical response of rat vertebrae is apparent through the application of image registration to analyse vertebral body behaviour; such information would not be evident in analysing preclinical whole vertebral body response using finite element modeling or experimental testing protocols.

Bone is the preferred site of metastases in women with breast cancer, which can cause skeletal-related events (SRE¡¦s) such as pathologic fractures. Bisphosphonates are the current standard of care for treatment of meta-static bone disease by preventing further bone destruction. Photodynamic therapy (PDT) has been applied successfully as a non-radiative treatment for malignancies. In PDT, light is delivered to a tumour after the administration of a photosensitiser. Earlier pre-clinical studies in a metastatic rat model have shown that PDT reduced the tumour burden in the vertebrae. The goal of this investigation was to study the effect of PDT on bisphosphonate pre-treated cancer in-vitro.

Human breast cancer cells, MT-1, were cultured until confluent. The following groups were formed: no treatment; incubation with zoledronic acid (24h; 10 ƒÝmol) only; PDT treatment only and incubation with zoledronic acid and PDT treatment. Prior to light application 1 microg/ml of the photosensitiser BPD-MA was added. PDT was performed with a light dose of 1J and 10 J. The cells were stained with a live/dead stain and analyzed by fluorescence microscope and flowcytometry.

Incubation of the MT-1 carcinoma cells with bisphosphonate zoledronic acid resulted in a significantly higher number of dying cells following PDT treatment when compared cells that were not treated by zoledronic acid (p< 0.05). When comparing cell groups that did not undergo PDT treatment the incubation with zoledronic acid alone did not have a statistically significant effect on cell survival twenty-four hours following zoledronic acid administration.

In-vitro, breast cancer cells appear more susceptible to PDT after they have been incubated with the zoledronic acid. Zoledronic acid, a potent bisphosphonate, inhibits farsenylpyrophosphate (FPP) which is involved in farsenylation of cell membrane proteins. The inhibition of FPP may cause a reduced effect of PDT on cell rescue. The treatment with bisphosphonates seems to have a synergistic effect with PDT treatment. As such, light dosimetry in PDT treatment may need to take into account potential therapeutic interactions between PDT and current medical therapies in the treatment of skeletal metastatic burden.

Versican is a large extracellular proteoglycan that is expressed in a variety of tissues and primary malignancies including infiltrating breast carcinoma. It also appears that versican can inhibit intercellular adhesion of normal as well as malignant cells. With the observation of selectin-like properties of versican G3 the investigators hypothesise that versican G3 influences not only local tumour invasiveness but also systemic metastases including the spread to bony sites. The present study aimed to test the hypothesis of versican G3 associated metastatic invasiveness in a murine osteolytic metastatic model of human breast carcinoma.

Human carcinoma cells (MT-1), transfected with the either a versican-G3-construct (n=7) or a vector-control (n=8) gene, were injected intracardically female athymic rats. The rats were examined clinically at serial time-points following injection and animal weight recorded. Animals were euthanised three weeks after tumour cell injection. On digital lateral radiographs of the scapula osteolytic areas were measured. Additionally, histomorphometry was performed on sections stained with human EGFr antibody to evaluate tumour burden within rodent vertebrae. Statistical analyses were performed using one way ANOVA.

All rats demonstrated weight loss approximately three weeks following tumour cell injection. However, the extent of weight loss observed over time was greater for the versicanG3 group (p< 0.05). Osteolytic metastases were observed using fine detail radiography at the day of euthanasia. Osteolytic burden was greater (p < 0.002) in the G3 transfected group (34.7 %; lytic area scapula) when compared to vector-control animals (8 %).

Versican G3 domain appears to influence the development of metastases to bone and soft tissue. The propensity of versican G3 to influence tumour invasion to bone and the mechanisms of versican G3 mediated osteolysis warrants ongoing study. With the known interactions between versican G3 and beta1 integrin in other cancer cell types and the increasing knowledge regarding several beta3 integrin-expressing cell populations, including osteoclasts in breast cancer tumour progression, the potential interaction between versican G3 and integrin receptors in bone may influence tumour mediating chemotactic and haptotactic migration towards bone factors.

Purpose: There is a clinical need for novel effective local therapies to treat spinal metastases at significant risk for fracture. Photodynamic therapy (PDT) is a promising cancer treatment that employs wavelength specific light combined with a photosensitizing agent to induce localized tumour destruction by photochemical generation of singlet oxygen. Using minimally invasive techniques developed for vertebroplasty to deliver light within the vertebral body, PDT is proposed as a potential new treatment for spinal metastases; however, the effects of PDT on bone are largely unknown. This study aims to determine if PDT affects the structural integrity of normal vertebral bone.

Methods: Sixteen Wistar rats were randomly assigned to control, 1-week treatment or 6-week treatment groups. Rats treated with PDT received an intracardiac injection of 2mg/kg BPD-MA activated at 15 minutes post-injection through administration of a non-thermal 690nm diode laser positioned adjacent to the L3 vertebral body via fluoroscopic guidance (150J at 150mW). Rats were sacrificed at 1-week or 6-weeks following a single treatment. |In vitro & #956;CT scans were taken of L2-L4 and 3D stereological quantities measured using a semiautomated volume shrinkage thresholding technique within the trabecular bone centrum. L2, L3 and L4 vertebral bodies were individually tested biomechanically to failure in axial compression. Yield stress and stiffness were calculated from generated load displacement curves.

Results: Bone surface area and bone volume significantly increased with treatment, through trabecular thickening, at both 1-week and 6-weeks vs. control group. The treated group demonstrated an increase in yield stress at 6-weeks vs. control (27%, p=0.023). An increase in stiffness (45%, p=0.010) was found in the 1-week treatment group vs. control, but was not maintained in the 6-week group.

Conclusions: PDT is a promising new treatment for spinal metastases that appears to strengthen vertebral bone. Further research must determine the mechanism of this action and verify if similar effects will occur in metastatically-involved vertebrae. If PDT proves to be effective in both destroying tumour cells and in strengthening remaining bone, it may provide a very attractive minimally invasive treatment option for patients with spinal metastases.

Funding : Other Education Grant

Funding Parties : Canadian Breast Cancer Foundation, Ontario Chapter