Primary bone tumors are rare, complex and highly heterogeneous. Its diagnostic and treatment are a challenge for the multidisciplinary team. Developments on tumor biomarkers, immunohistochemistry, histology, molecular, bioinformatics, and genetics are fundamental for an early diagnosis and identification of prognostic factors. The personalized medicine allows an effective patient tailored treatment. The bone biopsy is essential for diagnosis. Treatment may include systemic therapy and local therapy. Frequently, a limb salvage surgery includes wide resection and reconstruction with endoprosthesis, biological or composites. The risk for local recurrence and distant metastases depends on the primary tumor and treatment response.

Cancer patients are living longer and bone metastases are increasing. Bone is the third most frequently location for distant lesions. Bone metastases are associated to pain, pathological fractures, functional impairment, and neurological deficits. It impacts survival and patient quality of life. The treatment of metastatic disease is a challenge due to its complexity and heterogeneity, vascularization, reduced size and limited access. It requires a multidisciplinary treatment and depending on different factors it is palliative or curative-like treatment. For multiple bone metastases it is important to relief pain and increases function in order to provide the best quality of life and expect to prolong survival. Advances in nanotechnology, bioinformatics, and genomics, will increase biomarkers for early detection, prognosis, and targeted treatment effectiveness. We are taking the leap forward in precision medicine and personalized care.

Bone metastases radiographically appear as regions with high (i.e. blastic metastases) or low (i.e. lytic metastases) bone mineral density. The clinical assessment of metastatic features is based on computed tomography (CT) but it is still unclear if the actual size of the metastases can be accurately detected from the CT images and if the microstructure in regions surrounding the metastases is altered (Nägele et al., 2004, Calc Tiss Int). This study aims to evaluate (i) the capability of the CT in evaluating the metastases size and (ii) if metastases affect the bone microstructure around them.

Ten spine segments consisted of a vertebra with lytic or mixed metastases and an adjacent control (radiologically healthy) were obtained through an ethically approved donation program. The specimens were scanned with a clinical CT (AquilionOne, Toshiba: slice thickness:1mm, in-plane resolution:0.45mm) to assess clinical metastatic features and a micro-CT (VivaCT80, Scanco, isotropic voxel size:0.039mm) to evaluate the detailed microstructure. The volume of the metastases was measured from both CT and micro-CT images (Palanca et al., 2021, Bone) and compared with a linear regression. The microstructural alteration around the metastases was evaluated in the volume of interest (VOI) defined in the micro-CT images as the volume of the vertebral body excluding the metastases. Three 3D microstructural parameters were calculated in the VOI (CTAn, Bruker SkyScan): Bone Volume Fraction (BV/TV), Trabecular Thickness (Tb.Th.), Trabecular Spacing (Tb.Sp.). Medians of each parameter were compared (Kruskal-Wallis, p=0.05).

One specimen was excluded as it was not possible to define the size of the metastases in the CT scans. A strong correlation between the volume obtained from the CT and micro-CT images was found (R2=0.91, Slope=0.97, Intercept=2.55, RMSE=5.7%, MaxError=13.12%). The differences in BV/TV, Tb.Th. and Tb.Sp. among vertebrae with lytic and mixed metastases and control vertebrae were not statistically significant (p-value>0.6). Similar median values of BV/TV were found in vertebrae with lytic (13.2±2.4%) and mixed (12.8±9.8%) metastases, and in controls (13.0±10.1%). The median Tb.Th. was 176±18 ∓m, 179±43 ∓m and 167±91 ∓m in vertebrae with lytic and mixed metastases and control vertebrae, respectively. The median Tb. Sp. was 846±26 ∓m, 849±286 ∓m and 880±116 ∓m in vertebrae with lytic and mixed metastases and control vertebrae, respectively.

In conclusion, the size of vertebral metastases can be accurately assess using CT images. The 3D microstructural parameters measured were comparable with those reported in the literature for healthy vertebrae (Nägele et al., 2004, Calc Tiss Int, Sone et al., 2004, Bone) and showed how the microstructure of the bone tissue surrounding the lesion is not altered by the metastases.

Vertebral metastases are the most common type of malignant lesions of the spine. Although this tumour is still considered incurable and standard treatments are mainly palliative, the standard approach consists in surgical resection, which results in the formation of bone gaps. Hence, scaffolds, cements and/or implants are needed to fill the bone lacunae.

Here, we propose a novel approach to address spinal metastases recurrence, based on the use of anti-tumour metallic-based nanostructured coatings. Moreover, for the first time, a gradient microfluidic approach is proposed for the screening of nanostructured coatings having anti-tumoral effect, to determine the optimal concentration of the metallic compound that permits selective toxicity towards tumoral cells.

Coatings are based on Zinc as anti-tumour agent, which had been never explored before for treatment of bone metastases.

The customized gradient generating microfluidic chip was designed by Autodesk Inventor and fabricated from a microstructured mould by using replica moulding technique. Microstructured mould were obtained by micro-milling technique. The chip is composed of a system of microfluidic channels generating a gradient of 6 concentrations of drug and a compartment with multiple arrays of cell culture chambers, one for each drug concentration. The device is suitable for dynamic cultures and in-chip biological assays. The formation of a gradient was validated using a methylene blue solution and the cell loading was successful.

Preliminary biological data on 3D dynamic cultures of stromal cells (bone-marrow mesenchymal stem cells) and breast carcinoma cells (MDA-MB-231) were performed in a commercial microfluidic device.

Results showed that Zn eluates had a selective cytotoxic effect for tumoral cells. Indeed, cell migration and cell replication of treated tumoral cells was inhibited. Moreover, the three-dimensionality of the model strongly affected the efficacy of Zn eluates, as 2D preliminary experiments showed a high cytotoxic effect of Zn also for stromal cells, thus confirming that traditional screening tests on 2D cultured cells usually lead to an overestimation of drug efficacy and toxicity.

Based on preliminary data, the customized platform could be considered a major advancement in cancer drug screenings as it also allows the rapid and efficient screening of biomaterials having antitumor effect.

Decreasing the chance of local relapse or infection after surgical excision of bone metastases is a main goals in orthopedic oncology. Indeed, bone metastases have high incidence rate (up to 75%) and important cross-relations with infection and bone regeneration. Even in patients with advanced cancer, bone gaps resulting from tumor excision must be filled with bone substitutes. Functionalization of these substitutes with antitumor and antibacterial compounds could constitute a promising approach to overcome infection and tumor at one same time. Here, for the first time, we propose the use of nanostructured zinc-bone apatite coatings having antitumor and antimicrobial efficacy. The coatings are obtained by Ionized Jet Deposition from composite targets of zinc and bovine-derived bone apatite. Antibacterial and antibiofilm efficacy of the coatings is demonstrated in vitro against S. Aureus and E. Coli. Anti-tumor efficacy is investigated against MDA- MB-231 cells and biocompatibility is assessed on L929 and MSCs.

A microfluidic based approach is used to select the optimal concentration of zinc to be used to obtain antitumor efficacy and avoid cytotoxicity, exploiting a custom gradient generator microfluidic device, specifically designed for the experiments. Then, coatings capable of releasing the desired amount of active compounds are manufactured. Films morphology, composition and ion-release are studies by FEG- SEM/EDS, XRD and ICP. Efficacy and biocompatibility of the coatings are verified by investigating MDA, MSCs and L929 viability and morphology by Alamar Blue, Live/Dead Assay and FEG-SEM at different timepoints. Statistical analysis is performed by SPSS/PC + Statistics TM 25.0 software, one-way ANOVA and post-hoc Sheffe? test. Data are reported as Mean ± standard Deviation at a significance level of p <0.05.

Results and Discussion. Coatings have a nanostructured surface morphology and a composition mimicking the target. They permit sustained zinc release for over 14 days in medium. Thanks to these characteristics, they show high antibacterial ability (inhibition of bacteria viability and adhesion to substrate) against both the gram + and gram – strain.

The gradient generator microfluidic device permits a fine selection of the concentration of zinc to be used, with many potential perspectives for the design of biomaterials. For the first time, we show that zinc and zinc-based coatings have a selective efficacy against MDA cells. Upon mixing with bone apatite, the efficacy is maintained and cytotoxicity is avoided. For the first time, new antibacterial metal-based films are proposed for addressing bone metastases and infection at one same time. At the same time, a new approach is proposed for the design of the coatings, based on a microfluidic approach. We demonstrated the efficacy of Zn against the MDA-MB-231 cells, characterized for their ability to form bone metastases in vivo, and the possibility to use nanostructured metallic coatings against bone tumors. At the same time, we show that the gradient-generator approach is promising for the design of antitumor biomaterials. Efficacy of Zn films must be verified in vivo, but the dual-efficacy coatings appear promising for orthopedic applications.

Introduction

Patients (2.7M in EU) with positive cancer prognosis frequently develop metastases (≈1M) in their remaining lifetime. In 30-70% cases, metastases affect the spine, reducing the strength of the affected vertebrae. Fractures occur in ≈30% patients. Clinicians must choose between leaving the patient exposed to a high fracture risk (with dramatic consequences) and operating to stabilise the spine (exposing patients to unnecessary surgeries). Currently, surgeons rely on their sole experience. This often results in to under- or over-treatment. The standard-of-care are scoring systems (e.g. Spine Instability Neoplastic Score) based on medical images, with little consideration of the spine biomechanics, and of the structure of the vertebrae involved. Such scoring systems fail to provide clear indications in ≈60% patients.

The femur is a common site for skeletal bony metastases. The aim of this study is to evaluate the outcomes of femoral intramedullary nailing in prophylactic versus therapeutic treatment in femoral metastases.

All femoral nails between April 2011 and November 2015 at a district general hospital were assessed. Intramedullary nailing performed for prophylactic or therapeutic management were included. Outcomes include mortality, survival time and length of stay in hospital.

A total of 40 cases were included. In the prophylactic group there were 25 patients and in the therapeutic group there were 15 patients. In the prophylactic group, mean age was 70 years (range 41–91); male to female ratio is 23:17 and 26 patients of this group was deceased. In the therapeutic group, mean age was 76 years (range 56–92); male to female ratio 15:10 and 10 patients were deceased in this group. The most common primary was prostate carcinoma followed by breast carcinoma. In the prophylactic group, mean survival was 25 weeks (range 2–147) and in the therapeutic group mean survival was 20 weeks (range 2–39). The length of stay was 21 days (range 3–80) in the prophylactic group and 28 days (range 7–63) in the therapeutic group.

Femoral nailing for metastases helps improve quality of life and we observed a mean survival time of 20–25 weeks postoperatively in both therapeutic and prophylactic nailing.

Patients with cancer and bone metastases can have an increased risk of fracturing their femur. Treatment is based on the impending fracture risk: patients with a high fracture risk are considered for prophylactic surgery, whereas low fracture risk patients are treated conservatively with radiotherapy to decrease pain. Current clinical guidelines suggest to determine fracture risk based on axial cortical involvement of the lesion on conventional radiographs, but that appears to be difficult. Therefore, we developed a patient-specific finite element (FE) computer model that has shown to be able to predict fracture risk in an experimental setting and in patients. The goal of this study was to determine whether patient-specific finite element (FE) computer models are better at predicting fracture risk for femoral bone metastases compared to clinical assessments based on axial cortical involvement on conventional radiographs, as described in current clinical guidelines.

45 patients (50 affected femurs) affected with predominantly lytic bone metastases who were treated with palliative radiotherapy for pain were included. CT scans were made and patients were followed for six months to determine whether or not they fractured their femur. Non-linear isotropic FE models were created with the patient-specific geometry and bone density obtained from the CT scans. Subsequently, an axial load was simulated on the models mimicking stance. Failure loads normalized for bodyweight (BW) were calculated for each femur. High and low fracture risks were determined using a failure load of 7.5 × BW as a threshold. Experienced assessors measured axial cortical involvement on conventional radiographs. Following clinical guidelines, patients with lesions larger than 30 mm were identified as having a high fracture risk. FE predictions were compared to clinical assessments by means of diagnostic accuracy values (sensitivity, specificity and positive (PPV) and negative predictive values (NPV)).

Seven femurs (14%) fractured during follow-up. Median time to fracture was 8 weeks. FE models were better at predicting fracture risk in comparison to clinical assessments based on axial cortical involvement (sensitivity 100% vs. 86%, specificity 74% vs. 42%, PPV 39% vs. 19%, and NPV 100% vs. 95%, for the FE computer model vs. axial cortical involvement, respectively). We concluded that patient-specific FE computer models improve fracture risk predictions of femoral bone metastases in advanced cancer patients compared to clinical assessments based on axial cortical involvement, which is currently used in clinical guidelines. Therefore, we are initiating a pilot for clinical implementation of the FE model.

Background

A dedicated referral pathway for patients with bony metastases was introduced at Guy's and St Thomas’ Hospitals (GSTT) in 2009. The aim was to facilitate prompt, consultant-led decision-making and intervention for patients at risk of pathological fracture of long bones.

Background

Metastatic bone patients who require surgery needs to be evaluated in order to maximise quality of life and avoiding functional impairment, minimising the risks connected to the surgical procedures. The best surgical procedure needs to be tailored on survival estimation. There are no current available tool or method to evaluate survival estimation with accuracy in patients with bone metastasis. We recently developed a clinical decision support tool, capable of estimating the likelihood of survival at 3 and 12 months following surgery for patients with operable skeletal metastases. After making it publicly available on www.PATHFx.org, we attempted to externally validate it using independent, international data.