Residual tumor cells left in the bone defect after malignant bone tumor resection can result in local tumor recurrence and high mortality. Therefore, ideal bone filling materials should not only aid bone reconstruction or regeneration, but also exert local chemotherapeutic efficacy. However, common bone substitutes used in clinics are barely studied in research for local delivery of chemotherapeutic drugs. Here, we aimed to use facile manufacturing methods to render polymethylmethacrylate (PMMA) cement and ceramic granules suitable for local delivery of cisplatin to limit bone tumor recurrence. Porosity was introduced into PMMA cement by adding 1-4% carboxymethylcellulose (CMC) containing cisplatin, and chemotherapeutic activity was rendered to two types of granules via adsorption. Then, mechanical properties, porosity, morphology, drug release kinetics, ex vivo reconstructive properties of porous PMMA and in vitro anti-cancer efficacy against osteosarcoma cells were assessed. Morphologies, molecular structures, drug release profiles and in vitro cytostatic effects of two different drug-loaded granules on the proliferation of metastatic bone tumor cells were investigated. The mechanical strengths of PMMA-based cements were sufficient for tibia reconstruction at CMC contents lower than 4% (≤3%). The concentrations of released cisplatin (12.1% and 16.6% from PMMA with 3% and 4% CMC, respectively) were sufficient for killing of osteosarcoma cells, and the fraction of dead cells increased to 91.3% within 7 days. Functionalized xenogeneic granules released 29.5% of cisplatin, but synthetic CaP granules only released 1.4% of cisplatin over 28 days. The immobilized and released cisplatin retained its anti-cancer efficacy and showed dose-dependent cytostatic effects on the viability of metastatic bone tumor cells. Bone substitutes can be rendered therapeutically active for anticancer efficacy by functionalization with cisplatin. As such, our data suggest that multi-functional PMMA-based cements and cisplatin-loaded granules represent viable treatment options for filling bone defects after bone tumor resection

Infections are among the most diffused complications of the implantation of medical devices. In orthopedics, they pose severe societal and economic burden and interfere with the capability of the implants to integrate in the host bone, significantly increasing failure risk. Infection is particularly severe in the case of comorbidities and especially bone tumors, since oncologic patients are fragile, have higher infection rate and impaired osteoregenerative capabilities. For this reason, prevention of infection is to be preferred over treatment. This is even more important in the case of spine surgery, since spine is among the main site for tumor metastases and because incidence of post operative surgical-site infections is significant (up to 15-20%) and surgical options are limited by the need of avoiding damaging the spinal cord. Functionalization of the implant surfaces, so as to address infection and, possibly, co- adjuvate anti-tumor treatments, appears as a breakthrough innovation. Unmet clinical needs in infection and tumors is presented, with a specific focus on the spine, then, new perspectives are highlighted for their treatment

Functionalization of biomimetic nanomaterials allows to reproduce the composition of native bone, permitting better regeneration, while nanoscale surface morphologies provide cues for cell adhesion, proliferation and differentiation. Functionalization of 3D printed and bioprinted constructs, by plasma-assisted deposition of calcium phosphates-based (CaP) nanostructured coatings and by nanoparticles, respectively, will be presented. Stoichiometric and ion doped CaP- based nanocoatings, including green materials (mussel seashells and cuttlefish bone), will be introduced to guide tissue regeneration. We will show interactions between biomimetic surfaces and MSCs to address bone regeneration and SAOS-2 cells for bone tumor models. Our results show that combining AM and nanostructured biomimetic films permits to reproduce the architecture and the mechanical and compositional characteristics of bone. Stability behavior of the coatings, as well as MSCs behavior strongly depend on the starting CaP material, with more soluble CaPs and ion-doped ones showing better biological behavior. Green materials appear promising, as biomimetic films can be successfully obtained upon conversion of the marine precursors into hydroxyapatite. Last-not-least, nanoparticles-loaded scaffolds could be bioprinting without loss of cell viability, but ink characteristics depend on ion-doping as demonstrated for SAOS-2 cells over 14 days of culture. Biomimetic nanomaterials for functionalization in AM is a promising approach for bone modelling and regeneration

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

Extensive bone defects, caused by severe trauma or resection of large bone tumors, are difficult to treat. Regenerative medicine, including stem cell transplantation, may provide a novel solution for these intractable problems and improve the quality of life in affected patients. Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine due to their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. However, the osteogenic potential of ASCs is lower than that of bone marrow-derived stromal/stem cells. To address this disadvantage, our group has employed various methods to enhance osteogenic differentiation of ASCs, including factors such as bone morphogenetic protein or Vitamin D, coculture with bone marrow stem cells, VEGF transfection, and gene transfer of Runx-2 and osterix. Recently, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation while GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species and increased GSH/GSSG ratios were also detected in GSTT1- transfected ASCs. GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones

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

Summary Statement. Treatment of non-union is a highly demanding field with respect to bone healing. BMP 7 is a useful, wide-ranged tool in treating non-union of the foot and benign bone tumors. It represents a low-risk procedure with a high level of reliability. Introduction. Treatment of non-union is a highly demanding field with respect to bone healing. Treatment of tibial fracture non-union with the bone morphogenetic protein 7 (BMP-7) has been successfully reported. BMP 7 is a recombinant human protein produced in ovary cells of the Chinese hamster. It is responsible for the differentiation of mesenchymal stem cells from the periost, muscle and sponious bone and stimulates bone formation. It is the aim of our study to investigate the use of BMP 7 for other locations than the tibia, such as the foot and benign bone tumors. We strive for union or revision in each medical case. Patients & Methods. At our clinic we applied BMP-7 to 13 patients (9 patients with non-union, 4 patients with benign bone cysts). 9 patients with non-union of the foot (4 forefoot, 1 midfoot, 3 hindfoot, 1 tibia) were surgically treated by resection, stabilisation, and application of BMP 7. The study included 5 men and 4 women at an average age of 58,4 years (range 33 – 80), 13 previous surgeries had been carried out. The period of follow up was on average 16.3 months (5 – 40 months). The indication for using BMP-7 instead of autologous bone graft was poor local blood supply, poor local soft tissue because of previous interventions and risk factors like smoking and diabetes. Following an indicated open biopsy, the 4 cases of benign bone tumors (1 juvenile bone cyst of the talus, 1 osteofibrose dysplasia of the proximal tibia and 2 juvenile bone cysts of the proximal humerus) were all treated with resection, followed by an application of BMP-7 and external or internal fixation. In addition two received bone grafting and two received cortisone. The average age of the tumor group was 16,75 years (11–24 years, 2 male, 2 female). Results. At follow-up all patients were satisfied with respect to pain and function, no operative complications had occurred and bone fusion had finished in 7 patients after 3 months. One ankle joint had a fibrous fusion but was free of pain. One arthrodesis of the first metatarsophalangeal joint was turned into a resection arthroplasty, today the patient is free of pain and uses a normal shoe. Both bone cysts have the radiological evidence of rehabilitation. At one humeruscyst we removed the TENS-nails without complications. We had no complications like heterotopic ossification, local erythema or pressure sensitivity. Discussion/Conclusion. These results show that BMP 7 is a useful, wide-ranged tool in treating non-union of the foot and benign bone tumors. It represents a low-risk procedure with a high level of reliability

Background. In certain clinical situations, complex local anatomy and limitations of surgical exposure can make adequate and bone tumor ablation, resection and reconstruction very challenging. We wished to review our clinical experience and accuracy achieved with entirely virtually planned single stage tumor ablation/resection and reconstructions. Methods. We report 6 cases of bone tumors in which tumor removal (by radio-frequency (RF) ablation and/or resection) and subsequent reconstruction were based entirely on pre-operative virtual analysis and planning. All interventions were accomplished with specifically designed and pre-operatively manufactured 3D-printed drill & resection guides. Immediate subsequent defect reconstruction was either performed with a precisely matching allograft (n=1) or composite metal implant (n=5) consisting of a defect specific titanium scaffold and multiple integrated fixation features to provide optimal immediate stability as well as subsequent opportunity for osseointegration. We reviewed the sequence of all procedural steps as well as the accuracy of each saw blade or drill trajectory by direct intra-operative measurement, post-operative margin status and virtual comparison of pre- and post-operative CT scans. Results. Intra-operative application/assembly of the resection guides could be accomplished with relative ease in all cases, permitting quick and efficient reproduction of the planned osteotomies as well as RF-probe trajectories with a high degree of accuracy. Histologically all resection margins were negative as planned except in one case where one pelvic resection was extended due to intraoperative concern of possible local tumor progression. All implants could be placed as planned, with post-operative imaging demonstrating satisfactory implant position. Virtual analysis of post-operative CT scans confirmeded minimal deviation of final implant position from the pre-operative plan. Conclusion. Reliable, accurate placement of tumor biopsy/ablation tracts and resection planes and their optimal alignment with respect to critical structures, tumor extent and desired preservation of unaffected bone is the most challenging and time consuming step during the analysis and planning phase. However it is also the crucial step with regard to subsequent design and production of clinically and oncologically meaningful case-specific drill/resection guides and implants. If these prerequisites are met, computer assisted virtual planning along with 3Dprinting-technology can afford high intraoperative accuracy, contribute to increased intra-operative surgeon confidence and decreased operative time

The purpose of our study was to identify possible risk factors of patients with GCT of the long bones after curettage and packing the bone cavity with bone cement or bone allografts. We retrospectively reviewed the records of 249 patients with GCT of the limbs treated at Musculoskeletal Oncology Department of our institution between 1990 and 2013, confirmed histologically and recorded in the Bone Tumor Registry. We reviewed 219 cases located in the lower limb and 30 of the upper limb. This series includes 135 females and 114 males, with mean age 32 years (ranging 5 to 80 yrs). According to Campanacci's grading system, 190 cases were stage 2, 48 cases stage 3, and 11 cases stage 1. Treatment was curettage (intralesional surgery). Local adjuvants, such as phenol and cement, were used in 185 cases; whereas in the remaining 64 cases the residual cavity was filled with allografts or autografts only. Oncological outcome shows 203 patients alive and continuously disease-free (CDF), 41 patients NED1 after treatment of local recurrence (LR), 2 patients NED1 after treatment of lung metastases, 2 AWD with lung metastases. One patient died of unrelated causes (DOD). LR rate was 15.3% (38 pts). Lung metastases rate was 1.6% (4 pts). In patients treated by curettage and cement (185 cases) LR was 12% (22 pts). Conversely, in patients treated curettage and bone allografts it was higher (16/64 cases), with an incidence of 25% of cases (p=0.004). Oncological complications seemed to be related with site, more frequently occurring in the proximal femur (p=0.037). LR occurred only in stage 2 or 3 tumors without statistical significance (p>0.05). The mean interval between the first surgical treatment and LR was 22 months (range: 3–89 mos). However, in the multivariate analysis no significant statistical effect on local recurrence rate could be identified for gender, patient's age, Campanacci's grading, or cement vs allografts. The only independent risk factor related to the local recurrence was the site, with a statistical significance higher risk for patients with GCT of the proximal femur (p= 0.008). Our observation on the correlation of tumor location and risk of local recurrence is new. Therefore, special attention must be given to GCTs in the proximal femur. In fact, primary benign bone tumors in the proximal femur are difficult to treat due to the risk of secondary osteonecrosis of the femoral head or pathologic fracture. Numerous methods of reconstructions have been reported. Among these, total hip arthroplasty (THA) or bipolar hip arthroplasty (BHA) should be avoided when possible as more cases are observed in young patients. Therefore, we do not suggest different approach for the proximal femur. GCT in the proximal femur is much more difficult to treat than in other sites, but if curettage is feasible, the best way is to save the joint with a higher risk of local recurrence, knowing that the sacrifice of the hip articulation in case of recurrence is always possible with THA or BHA

The initiation and progression of malignant tumors are supported by their microenvironment: cancer cells per se cannot explain growth and formation of the primary or metastasis, and a combination of proliferating tumor cells, cancer stem cells, immune cells, mesenchymal stromal cells and/or cancer-associated fibroblasts all contribute to the tumor bulk. The interaction between these multiple players, under different microenvironmental conditions of biochemical and physical stimuli (i.e. oxygen tension, pH, matrix mechanics), regulates the production and biological activity of several soluble factors, extracellular matrix components, and extracellular vesicles that are needed for growth, maintenance, chemoresistance and metastatization of cancer. Both in osteosarcoma and bone metastases from carcinomas this aspect has been only recently explored. In this lecture, I will discuss the role of tumor microenvironment, with a particular focus on the mesenchymal stroma, contributing to bone tumor progression through inherent. The most recent advances in the molecular cues triggered by cytokines, soluble factors, and metabolites that are partially beginning to unravel the axis between stromal elements of mesenchymal origin and bone cancer cells, under different microenvironmental conditions, will be reviewed providing insights likely to be used for novel therapeutic approaches

Biomechanical interpretations of bone adaptation in biological reconstructions following bone tumors would be crucial for orthopedic oncologists, particularly if based on quantitative observations. This would help to plan for surgical treatments, rehabilitative programs and communication with the patients. In particular, outcomes of the Capanna technique, which combines bone allograft and vascularized fibula autograft, lead to stable and durable reconstructions [1, 2], and different remodeling patterns have been described [3] as a response to mechanical loading. However, there are several events that are not understood and require a biomechanical interpretation, as the evolution patterns can evolve towards conditions that threaten the strength of the reconstruction. We aimed to (i) analyze the biomechanical adaptation of a femoral reconstruction after Ewing sarcoma, in terms of morphological and densitometric evolution of bone from CT data, internal loads acting on the bone during movement, mechanical competence of the reconstruction, and (ii) relate in-progress bone resorption to the mechanical stimulus induced by different motor activities. Eight CT datasets of a patient (8 yrs at surgery using the Capanna technique) during 76-month follow-up were available. The evolution of bone morphology, density and moments of inertia was quantified. At the last control, the patient underwent gait analysis (walking, chair rise/sit, stair ascent/descent, squat). We created a multiscale musculoskeletal and finite element model from CT scans and motion analysis data at the end of follow-up, using state-of-the-art modeling workflows [4, 5], to analyze muscle and joint loads, and to compare the mechanical competence of the reconstructed bone with the contralateral limb, in the current real condition and in a possible revision surgery that removed proximal screws. Although there were no reconstruction complications and osteo-fusion with intense remodeling between allograft and autograft was shown, there was a progressive decrease in allograft cortical thickness and density. There were strategies of muscle coordination that led to differences in joint loads between limbs more marked in more demanding motor activities, and generally larger in the contralateral limb. The operated femur presented a markedly low ratio of physiological strain due to load-sharing with the metal implant, particularly in the lateral aspect. A possible revision surgery removing the three most proximal screws would help restore a physiological strain configuration, while the safety of the reconstruction would not be threatened. We suggest that bone resorption is related to load-sharing and to the internal forces exerted during movement, and the mechanical stimulus should be improved by adopting modifications in the surgical treatment and by promoting physical therapy aimed at specific muscle strengthening

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. Introduction. Use of radiofrequency ablation (RFA) in treating of skeletal metastases has been rising, yet its impact on bone tissue is poorly understood. 2–11 RF treatment induces frictional heating and effectively necrotises tissue in a local and minimally invasive manner.1 Bipolar cooled RF (BCRF) is a significant improvement to conventional RF whereby larger regions can be safely treated, protecting sensitive neighbouring tissues from thermal effects. This study aimed to evaluate the safety and feasibility of a novel bipolar RFA probe to create large contained lesions within healthy pig vertebrae and its determine its effects on bone and tumour cells in a rabbit long bone tumour model. Methods. Following a pre-treatment MRI, a BCRF probe was placed transpedicularly into targeted lumbar vertebrae of six Yorkshire pigs. Energy was delivered for 15min at a set temperature of 65°C (n=2 per animal) with a sham control performed at a non-contiguous level (n=1 per animal). Post-treatment neurologic evaluation, MRI and histology were used to characterise the region of effect. Twelve New Zealand White Rabbits received a 200 µl injection of VX2 tumour cells into one femur. On day 14, half of the tumour-bearing and contralateral healthy femora were RF-treated (n=6 per group). RF-treated femora were compared to tumour-bearing and healthy sham groups (n=6 per group) through pre (day 14) and post treatment (day 28) MRI and histology (H&E (for general evaluation), AE1/AE3 (for VX2 tumour cell evaluation), TRAP (for osteoclast evaluation) and TUNEL (for osteocyte evaluation)). Results. In treated porcine spines there were no neurological complications. MR imaging confirmed a 2cm oval shaped ablative zone. External thermocouple measurements indicated output values in the physiological temperature range suggesting treatment was safely confined within targeted vertebrae. Histological results correlated well with the ablation regions determined using MRI sequences in both models. In rabbit femora, large zones of RF ablation (average volume 12.9±5.5 cm3) extended beyond the femur cortex (corresponding to the probe design for human use) into the surrounding soft tissue. The RFA-treated tumour-involved specimens demonstrated a significant reduction in tumour volume compared to sham femora, however a small number of viable tumour cells remained within the ablation volume. Newly formed trabecular structures were also seen in all treated femora. TRAP staining demonstrated a significant reduction in osteoclast number post-RFA in both the tumour-involved and healthy groups. TUNEL staining revealed areas of patchy cortical osteocyte necrosis within the ablation zone. Discussion/Conclusions. The large histologic region of effect created by RFA was consistent with MRI findings in both models. Treatment was contained in the porcine vertebrae without collateral damage to neighbouring sensitive structures. In the femora, while osteoclasts were found to be very susceptible to RFA, a small number of tumour cells and osteocytes in the treated regions remained viable. As the treatment zone did not encompass the full extent of the intramedullary lesions, it is possible that the sporadic VX2 cell viability may be explained by local tumour cell migration. Limited destruction of healthy osteocytes by RFA may be desirable in restoring bone health

Objectives

We evaluated the accuracy of augmented reality (AR)-based navigation assistance through simulation of bone tumours in a pig femur model.