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Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_1 | Pages 43 - 43
2 Jan 2024
Lipreri M Cortini M Baldini N Avnet S
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Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research.

Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms.

To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode.


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_1 | Pages 16 - 16
2 Jan 2024
Lipreri M Pasquarelli A Scelfo D Baldini N Avnet S
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Osteoporosis is a progressive, chronic disease of bone metabolism, characterized by decreased bone mass and mineral density, predisposing individuals to an increased risk of fractures. The use of animal models, which is the gold standard for the screening of anti-osteoporosis drugs, raises numerous ethical concerns and is highly debated because the composition and structure of animal bones is very different from human bones. In addition, there is currently a poor translation of pre-clinical efficacy in animal models to human trials, meaning that there is a need for an alternative method of screening and evaluating new therapeutics for metabolic bone disorders, in vitro.

The aim of this project is to develop a 3D Bone-On-A-Chip that summarizes the spatial orientation and mutual influences of the key cellular components of bone tissue, in a citrate and hydroxyapatite-enriched 3D matrix, acting as a 3D model of osteoporosis. To this purpose, a polydimethylsiloxane microfluidic device was developed by CAD modelling, stereolithography and replica molding. The device is composed by two layers: (i) a bottom layer for a 3D culture of osteocytes embedded in an osteomimetic collagen-enriched matrigel matrix with citrate-doped hydroxyapatite nanocrystals, and (ii) a upper layer for a 2D perfused co-culture of osteoblasts and osteoclasts seeded on a microporous PET membrane.

Cell vitality was evaluated via live/dead assay. Bone deposition and bone resorption was analysed respectively with ALP, Alizarin RED and TRACP staining. Osteocytes dendrite expression was evaluated via immunofluorescence. Subsequently, the model was validated as drug screening platform inducing osteocytes apoptosis and administrating standard anti-osteoporotic drugs.

This device has the potential to substitute or minimize animal models in pre-clinical studies of osteoporosis, contributing to pave the way for a more precise and punctual personalized treatment.


Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_14 | Pages 40 - 40
1 Dec 2022
Lipreri M Vecchione R Corrado B Avnet S Perut F Baldini N Graziani G
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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.