Osteosarcoma (OS) is a highly malignant primary tumor frequently occurring in children and adolescents. The mainstay of therapy is neoadjuvant chemotherapy and surgical removal of the lesion yielding a 50–70% of 5-year survival rate. Unfortunately, chemotherapy is currently unable to induce complete tumor necrosis leaving residual tumor cells free to metastasize or recidivate, thus resulting in a 30% mortality. The major limitation in those patients is the development of multidrug resistance (MDR) and the low water solubility of drugs such as Paclitaxel (PTX) that is in fact not included in the majority of chemotherapy protocols for OS treatment.

We thus hypothesized to prevent the emergence of MDR and obtain significant tumor reduction, by engineering innovative nanoparticles (NPs) able to vehiculate the PTX and induce a dual synergic action: the cytostatic effect of PTX and the cytotoxicity generated by reactive oxygen species produced from light triggered photoactivation (PDT) of Chlorin e6 photosensitizer. To further improve the efficacy and reduce the side effects of NPs systemic administration, Mesenchymal Stromal Cells (MSC) are used as a “Trojan horse” to deliver the NPs directly to tumor cells, taking advantage of MSC ability to selectively recognize and efficiently engraft in OS tumor stroma.

HSA were conjugated with photosensitizer Ce6 and the functionalized protein was used to produce PTX loaded nanoparticles through desolvation technique and drug-induced protein self-assembly (PTX-Ce6@HSA NP).

Human MSC lines, isolated from the Bone marrow (BM) of different donors, were then loaded with different dosages of nanoparticles and their ability to internalize and transport the NPs, migrate and induce cytotoxic ROS upon light treatment were tested in in vitro cultures.

Preliminary results showed that MSC efficiently internalize PTX-Ce6@HSA NPs and the photosensitizer Ce6 remains active inside the cells for at least 3 days after loading.

Electron microscopy performed onto loaded MSC showed that NPs internalization take places via clathrin mediated transport, whereas HPLC analysis demonstrated a release kinetics of PTX mediated by exocytosis. Finally, PTX-Ce6@HSA NPs loaded MSC co-cultured with the OS tumor cell line SaOS-2 showed a significant tumor cell growth reduction.

So fare, advances in drug delivery have failed to produce specific tool to improve the overall survival of OS patients. However, given our preliminary in vitro data we believe that the proposed multimodal therapy will minimize the side effects of the systemic chemotherapy and enhance the efficacy through the synergic effect of PTX and PDT.

In the future, our strategy could be intended as an innovative co-adjuvant approach for OS treatment to be performed right before surgery to eliminate residual tumors cells after tumor mass removal.

Demineralized bone matrix (DBM) is a natural, collagen-based, well-established osteoinductive biomaterial. Nevertheless, there are conflicting reports on the efficacy of this product. The purpose of this study was to evaluate whether DBM collagen structure is affected by particle size and can influence DBM osteoinductivity.

Sheep cortical bone was ground and particles were divided in three fractions with different sizes, defined as large (L, 1–2 mm), medium (M, 0.5–1 mm), and small (S, < 0.5 mm). After demineralization, the three DBM samples were characterized by DTA analysis, XRD, ICP-OES, and FTIR. Data clearly showed a particle size-dependent alteration in collagen structure, with DBM-M being altered but not as much as DBM-S. The in vivo study showed that only DBM-M was able to induce new bone formation in a subcutaneous ectopic mouse model. When sheep MSC were seeded onto DBM particles before implantation, all DBM particles were able to induce new bone formation with the best incidence for DBM-M and DBM-S. Gene expression analysis performed on recovered implants supports the histological results and underlines the supportive role of MSC in DBM osteoinduction through the regulation of host cells. In conclusion, our results show a relation between DBM particle size, structural modification of the collagen and in vivo osteoinductivity. The medium particles represent a good compromise between no modification (largest particles) and excessive modification (smallest particles) of collagen structure, yielding highest osteoinduction. We believe that these results can guide researchers to use DBM particles of 0.5–1 mm size range in applications aimed at inducing new bone formation, obtaining results more comparable and reliable among different research groups. Furthermore, we suggest to carefully analyze the structure of the collagen when a collagen-based biomaterial is used alone or in association with cells to induce new bone formation.

Introduction

The delay looks radiographically as a fracture callus not very evident or absent 6 months after osteosynthesis. Patients undergo a long period of immobilization and this fact causes the increase the social cost of the disease. The technique we suggest aims to the reduce the period of immobilization and as a consequence the management costs of the disease.