Various chemicals are commonly used as adjuvant treatment to surgery for giant-cell tumour (GCT) of bone. The comparative effect of these solutions on the cells of GCT is not known. In this study we evaluated the cytotoxic effect of sterile water, 95% ethanol, 5% phenol, 3% hydrogen peroxide (H. 2. O. 2. ) and 50% zinc chloride (ZnCI. 2. ) on GCT monolayer tumour cultures which were established from six patients. The DNA content, the metabolic activity and the viability of the cultured samples of tumour cells were assessed at various times up to 120 hours after their exposure to these solutions. Equal cytotoxicity to the GCT monolayer culture was observed for 95% ethanol, 5% phenol, 3% H. 2. O. 2. and 50% ZnCI. 2. The treated samples showed significant reductions in DNA content and metabolic activity 24 hours after treatment and this was sustained for up to 120 hours. The samples treated with sterile water showed an initial decline in DNA content and viability 24 hours after treatment, but the surviving cells were viable and had proliferated. No multinucleated cell formation was seen in these cultures. These results suggest that the use of chemical adjuvants other than water could help improve local control in the treatment of GCT of bone

Summary. We demonstrate that osteoclast-like cells of GCT result from the spontaneous fusion and differentiation of CD14+ cells of the monoblastic lineage by an autocrine mechanism mediated by RANKL, rather than induced by stromal cells. This process is further enhanced by the simultaneous impairment of the negative feed-back regulation of osteoclastogenesis by interferon β. Introduction. Giant cell tumor of bone (GCT) is a benign osteolytic lesion with a complex histology, comprising prominent multinucleated osteoclast-like cells (OC), mononuclear stromal cells (SC), and monocyte-like elements. So far, most studies have focused on SC as the truly transformed elements that sustain osteoclast differentiation, while less attention has been paid on the monocyte-like cell fraction. On the contrary, we have previously shown that SC are non-transformed element that can induce osteoclastogenesis of monocytes at levels that do not exceed that of normal mesenchymal stromal cells. We therefore focused on CD14+ monocyte-like cells as an alternative key candidate for the pathogenesis of GCT. Methods. We isolated CD14+ enriched cell fraction from tumor samples by immunomagnetic separation. We analyzed CD14+ cells for ultrastructural morphology, mRNA levels of haematopoietic, monocytic, and dendritic markers, and for RANKL, and M-CSF. Due to the very high number of OC in GCT, we hypothesised that the IFN-b pathway might be impaired. In fact, IFN-b functions as a negative-feedback regulator that inhibits osteoclast differentiation. We assayed IFN-b mRNA and protein expression in both cultures and tumor samples. Finally, we verified the ability of CD14+ cells to spontaneously form osteoclasts. Results. In the CD14+ enriched fraction we identified two different cell populations, both positive for TRACP activity and negative for Ki-67 nuclear localization, one with an undefined histotype and the other showing characteristics of the monoblastic lineage, mainly monoblasts and promonocytes. Isolated cells were positive for CD45, MSE-1, RANK, CD14, and CD80, and negative for CD144, and were able to spontaneously form collagen-resorbing multinucleated cells, a process that was strongly impaired by the addition of osteoprotegerin. The expression of RANKL and M-CSF mRNA in cultured cells demonstrated the presence of an autocrine circuit inducing osteoclast formation. Finally, we found very low expression of IFN-b both in the in vitro formed OC and in tissue samples. Conclusions. These data show that CD14+ cells in GCT are monocyte-like cells that can spontaneously form bone-resorbing multinucleated cells through impaired IFN-b expression. Taken together, these data raise questions regarding the role of the CD14+ cell component and of their regulating mechanisms that may be relevant for the development of effective therapeutic strategies

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

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

Breast cancer is the most frequent malignancy in women with an estimation of 2.1 million new diagnoses in 2018. Even though primary tumours are usually efficiently removed by surgery, 20–40% of patients will develop metastases in distant organs. Bone is one of the most frequent site of metastases from advanced breast cancer, accounting from 55 to 58% of all metastases. Currently, none of the therapeutic strategies used to manage breast cancer bone metastasis are really curative. Tailoring a suitable model to study and evaluate the disease pathophysiology and novel advanced therapies is one of the major challenges that will predict more effectively and efficiently the clinical response. Preclinical traditional models have been largely used as they can provide standardization and simplicity, moreover, further advancements have been made with 3D cultures, by spheroids and artificial matrices, patient derived xenografts and microfluidics. Despite these models recapitulate numerous aspects of tumour complexity, they do not completely mimic the clinical native microenvironment. Thus, to fulfil this need, in our study we developed a new, advanced and alternative model of human breast cancer bone metastasis as potential biologic assay for cancer research. The study involved breast cancer bone metastasis samples obtained from three female patients undergoing wide spinal decompression and stabilization through a posterior approach. Samples were cultured in a TubeSpin Bioreactor on a rolling apparatus under hypoxic conditions at time 0 and for up to 40 days and evaluated for viability by the Alamar Blue test, gene expression profile, histology and immunohistochemistry. Results showed the maintenance and preservation, at time 0 and after 40 days of culture, of the tissue viability, biological activity, as well as molecular markers, i.e. several key genes involved in the complex interactions between the tumour cells and bone able to drive cancer progression, cancer aggressiveness and metastasis to bone. A good tis sue morphological and microarchitectural preservation with the presence of lacunar osteolysis, fragmented trabeculae locally surrounded by osteoclast cells and malignant cells and an intense infiltration by tumour cells in bone marrow compartment in all examined samples. Histomorphometrical data on the levels of bone resorption and bone apposition parameters remained constant between T0 and T40 for all analysed patients. Additionally, immunohistochemistry showed homogeneous expression and location of CDH1, CDH2, KRT8, KRT18, Ki67, CASP3, ESR1, CD8 and CD68 between T0 and T40, thus further confirming the invasive behaviour of breast cancer cells and indicating the maintaining of the metastatic microenvironment. The novel tissue culture, set-up in this study, has significant advantages in comparison to the pre-existent 3D models: the tumour environment is the same of the clinical scenario, including all cell types as well as the native extracellular matrix; it can be quickly set-up employing only small samples of breast cancer bone metastasis tissue in a simple, ethically correct and cost-effective manner; it bypasses and/or decreases the necessity to use more complex preclinical model, thus reducing the ethical burden following the guiding principles aimed at replacing/reducing/refining (3R) animal use and their suffering for scientific purposes; it can allow the study of the interactions within the breast cancer bone metastasis tissue over a relatively long period of up to 40 days, preserving the tumour morphology and architecture and allowing also the evaluation of different biological factors, parameters and activities. Therefore, the study provides for the first time the feasibility and rationale for the use of a human-derived advanced alternative model for cancer research and testing of drugs and innovative strategies, taking into account patient individual characteristics and specific tumour subtypes so predicting patient specific responses

Critical-sized bone defects can result from trauma, inflammation, and tumor resection. Such bone defects, often have irregular shapes, resulting in the need for new technologies to produce suitable implants. Bioprinting is an additive manufacturing method to create complex and individualised bone constructs, which can already include vital cells. In this study, we established an extrusion-based printing technology to produce osteoinductive scaffolds based on polycaprolactone (PCL) combined with calcium phosphate, which is known to induce osteogenic differentiation of stem cells. The model was created in python based on the signed distance functions. The shape of the 3D model is a ring with a diameter of 20 mm and a height of 10 mm with a spongiosa-like structure. The interconnected irregular pores have a diameter of 2 mm +/− 0.2 mm standard deviation. Extrusion-based printing was performed using the BIO X6. To produce the bioink, PCL (80 kDa) was combined with calcium phosphate nanopowder (> 150 nm particle size) under heating. After printing, 5 × 10. 6. hMSC were seeded on the construct using a rotating incubator. We were able to print a highly accurate ring construct with an interconnected pore structure. The PCL combined with calcium phosphate particles resulted in a precise printed construct, which corresponded to the 3D model. The bioink containing calcium phosphate nanoparticles had a higher printing accuracy compared to PCL alone. We found that hMSC cultured on the construct settled in close proximity to the calcium phosphate particles. The hMSC were vital for 22 days on the construct as demonstrated by life/dead staining. The extrusion printing technology enables to print a mechanically stable construct with a spongiosa-like structure. The porous PCL ring could serve as an outer matrix for implants, providing the construct the stability of natural bone. To extend this technology and to improve the implant properties, a biologised inner structure will be integrated into the scaffold in the future

The in vitro mimicking of bone microenvironment for the study of pathologies is a challenging field that requires the design of scaffolds with suitable morphological, structural and cytocompatible properties. During last years, 3D in vitro tumour models have been developed to reproduce mechanical, biochemical and structural bone microenvironment elements, allowing cells to behave as in vivo. In this work, gas foamed polyether urethane foams (PUF) and 3D printed thermoplastic polyether urethane (3DP-PU) designed with different patterns are proposed as scaffolds for in vitro model of bone tissue. Surface coatings for a biomimetic behaviour of the 3D scaffold models were also investigated. Morphological, chemico-physical, mechanical properties, and biological in vitro behaviour were investigated. PUFs for metastases investigation. The suitability of PUF as 3D in vitro model to study the interactions between bone tumour initiating cells and the bone microenvironment was investigated. PUF open porosity (>70%) appeared suitable to mimic trabecular bone structure. Human adipose derived stem cells (ADSC) were cultured and differentiated into osteoblast lineage on the PU foam, as confirmed by Alizarin Red staining and RT-PCR, thus offering a bone biomimetic microenvironment to the further co-culture with bone derived tumour-initiating cells (MCFS). Tumour aggregates were observed after three weeks of co-culture by e-cadherin staining and SEM; modification in CaP distribution was identified by SEM-EDX and associated to the presence of tumour cells. 3DP-PU as tumour bone model. 3D printed scaffolds have pores with a precise and regular geometry (0°-90°, 0°-45°-90°-135°, 0°-60°-120°). PU scaffold porosity evidenced values from 55 to 67%, values that belong to the porosity range of the trabecular bone tissue (30-90%). The compressive modulus varied between 2 and 4 MPa, depending on the printed pattern. Biomimetic nanostructured coating was performed on 0-90° 3DP-PU by Ionized Jet Deposition. Coatings had a submicrometric thickness, variable tuning deposition time, nanostructured surface morphology and biomimetic composition. Coating on 3DP-PU promoted cells colonization of the whole porous scaffolds, compared to the controls, where cells concentrated mostly on the outer layers. In conclusion, based on the obtained results, scaffolds with different geometries have been successfully produced. Morphological and structural properties of the scaffolds here presented are suitable for mimicking the bone tissue, in order to produce a 3D in vitro model useful for bone pathologies research

The cellular mechanisms which account for the formation of osteoclasts and bone resorption associated with enlarging benign and malignant mesenchymal tumours of bone are uncertain. Osteoclasts are marrow-derived, multinucleated, bone-resorbing cells which express a macrophage phenotype. We have determined whether tumour-associated macrophages (TAMs) isolated from benign and malignant mesenchymal tumours are capable of differentiating into osteoclasts. Macrophages were cultured on both coverslips and dentine slices for up to 21 days with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D. 3. (1,25(OH). 2. D. 3. ) and human macrophage colony-stimulating factor (M-CSF) or, in the absence of UMR 106 cells, with M-CSF and RANK ligand. In all tumours, the formation of osteoclasts from CD14-positive macrophages was shown by the formation of tartrate-resistant-acid-phosphatase and vitronectin-receptor-positive multinucleated cells which were capable of carrying out lacunar resorption. These results indicate that the tumour osteolysis associated with the growth of mesenchymal tumours in bone is likely to be due in part to the differentiation of mononuclear phagocyte osteoclast precursors which are present in the TAM population of these lesions

Abundant implant-derived biomaterial wear particles are generated in aseptic loosening and are deposited in periprosthetic tissues in which they are phagocytosed by mononuclear and multinucleated macrophage-like cells. It has been stated that the multinucleated cells which contain wear particles are not bone-resorbing osteoclasts. To investigate the validity of this claim we isolated human osteoclasts from giant-cell tumours of bone and rat osteoclasts from long bones. These were cultured on glass coverslips and on cortical bone slices in the presence of particles of latex, PMMA and titanium. Osteoclast phagocytosis of these particle types was shown by light microscopy, energy-dispersive X-ray analysis and SEM. Giant cells containing phagocytosed particles were seen to be associated with the formation of resorption lacunae. Osteoclasts containing particles were also calcitonin-receptor-positive and showed an inhibitory response to calcitonin. Our findings demonstrate that osteoclasts are capable of phagocytosing particles of a wide range of size, including particles of polymeric and metallic bio-materials found in periprosthetic tissues, and that after particle phagocytosis, they remain fully functional, hormone-responsive, bone-resorbing cells

Curettage and packing with polymethylmethacrylate cement is a routine treatment for giant-cell tumour (GCT) of bone. We performed an in vitro evaluation of the cytotoxic effect of a combination of cement and methotrexate, doxorubicin and cisplatin on primary cell cultures of stromal GCT cells obtained from five patients. Cement cylinders containing four different concentrations of each drug were prepared, and the effect of the eluted drugs was examined at three different time intervals. We found that the cytotoxic effect of eluted drugs depended on their concentration and the time interval, with even the lowest dose of each drug demonstrating an acceptable rate of cytotoxicity. Even in low doses, cytotoxic drugs mixed with polymethylmethacrylate cement could therefore be considered as effective local adjuvant treatment for GCTs

Local treatment with phenol is often used after intralesional excision of chondroblastomas and giant-cell tumours which involve bone near joints, and has been shown to reduce the rate of recurrence. The ideal concentration of phenol is uncertain, but may be important because of the high rate of absorption and toxicity. We have studied the effectiveness of different concentrations on standard sarcoma cell lines. Our results suggest that a 5% solution of phenol is effective against dispersed single cells, and that higher concentrations give no significant advantage, but create problems due to lack of homogeneous mixing, temperature and safety

Cancer-induced bone diseases are often associated with increased bone resorption and pathological fractures. In recent years, osteoprotective agents such as bisphosphonates have been studied extensively and have been shown to inhibit cancer-related bone resorption in experimental and clinical studies. The third-generation bisphosphonate, ibandronate (BM 21.0955), is a potent compound for controlling tumour osteolysis and hypercalcaemia in rats bearing Walker 256 carcinosarcoma. We have studied the effect of ibandronate given as an interventional treatment on bone strength and bone loss after the onset of tumour growth in bone. Our results suggest that it is capable of preserving bone quality in rats bearing Walker 256 carcinosarcoma cells. Since other bisphosphonates have produced comparable results in man after their success in the Walker 256 animal models our findings suggest that ibandronate may be a powerful treatment for maintaining skeletal integrity in patients with metastatic bone disease

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

Objectives

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

We undertook a study of the anti-tumour effects of hyperthermia, delivered via magnetite cationic liposomes (MCLs), on local tumours and lung metastases in a mouse model of osteosarcoma. MCLs were injected into subcutaneous osteosarcomas (LM8) and subjected to an alternating magnetic field which induced a heating effect in MCLs. A control group of mice with tumours received MCLs but were not exposed to an AMF. A further group of mice with tumours were exposed to an AMF but had not been treated with MCLs. The distribution of MCLs and local and lung metastases was evaluated histologically. The weight and volume of local tumours and the number of lung metastases were determined. Expression of heat shock protein 70 was evaluated immunohistologically. Hyperthermia using MCLs effectively heated the targeted tumour to 45°C. The mean weight of the local tumour was significantly suppressed in the hyperthermia group (p = 0.013). The mice subjected to hyperthermia had significantly fewer lung metastases than the control mice (p = 0.005). Heat shock protein 70 was expressed in tumours treated with hyperthermia, but was not found in those tumours not exposed to hyperthermia.

The results demonstrate a significant effect of hyperthermia on local tumours and reduces their potential to metastasise to the lung.