Controlled delivery of anticancer drugs through osteotropic nanoparticles (NP) is a novel approach for the adjuvant therapy of osteolytic bone metastases. Doxorubicin (DXR) is widely used in chemotherapy, although its activity is restricted by dose-dependent cardiotoxicity and marrow toxicity. However, its efficacy can be improved when specific targeting at the tumor site is obtained. The aim of this study was to obtain osteotropic biodegradable NP by nanoprecipitation of a copolymer between poly(D,L-lactide-co-glycolide) (PLGA) and an osteotropic bisphosphonate, sodium alendronate (ALE). NP were subsequently characterised for their chemical-physical properties, biocompatibility, and the ability to inhibit osteoclast-mediated bone resorption, and then loaded with DXR. The effectiveness of NP-loaded DXR was investigated through in vitro and in vivo experiments, and compared to that of free DXR. For the in vitro analysis, six human cell lines were used as a representative panel of bone tumors, including breast and renal adenocarcinoma, osteosarcoma and neuroblastoma. The in vitro uptake and the inhibition of tumor cell proliferation were verified. To analyse the in vivo activity of NP-loaded DXR, osteolytic bone metastases were induced through the intratibial inoculation in BALB/c-nu/nu mice of a human breast cancer cell line, followed by the intraperitoneal administration of the free or NP-loaded DXR. In vitro, aAll of the cell lines were able to uptake both free and NP-loaded drug, and their proliferation was inhibited up to 80% after incubation either with free or NP-loaded DXR. In addition, in vivo experiments showed that NP-loaded DXR were also able to reduce the incidence of bone metastases, not only in comparison with untreated mice, but also with free DXR-treated mice. In conclusion, this research demonstrated an improvement in the therapeutic effect of the antineoplastic drug DXR, when loaded to bone-targeted NP conjugated with ALE. Osteotropic PLGA-ALE NP are suitable to be loaded with DXR and offer as a valuable tool for a tissue specific treatment of skeletal metastases.

The effector function of natural killer (NK) cells is regulated by activating and inhibitory receptors, termed killer immunoglobulin-like receptors (KIRs). In haploidentical T-cell depleted transplantation the donor/recipient KIR mismatch significantly impacts on NK-mediated tumor cell killing, particularly in acute myeloid leukaemia (AML). Thirty-four high risk AML patients entered a phase I-II study of adoptive NK-cell based immunotherapy and were screened for the availability of one haploidentical KIR ligand mismatched donor. Thirteen of them resulted as having one suitable donor. NK cells were enriched from steady-state leukaphereses by using a double-step immunomagnetic separation system, consisting in depletion of CD3+ T cells followed by positive selection of CD56+ NK cells. CD56+ cells were enriched from 7,70% (1,26-11,70) to 93,50% (66,41-99,20) (median recovery 53,05% (30,97-72,85), median T-depletion 3,03 log (2,15-4,52) viability >92%) and their citotoxic activity was inalterate. All patients (4 progressions, 1 partial remission and 8 complete remissions) received NK cell infusion which was preceeded by immunosuppressive chemotherapy (fludarabine and cyclophosphamide) and followed by interleukin 2 injections. The median number of reinfused NK cells was 2,74x10(e)6/kg(1,11-5,00) and contamining CD3+ T cells were always less than 1x10(e)5/kg. The procedure was well-tolerated and no significant toxicity, including GvHD, related to NK cell infusion was observed. The donor NK cells were demonstrated in 5/10 patients. Among the 8 patients in complete remission 5 patients are stable after 18, 15, 4, 2 months of follow-up. Three other patients relapsed after 2 and 7 months. The patient in partial remission obtained a complete remission, which lasted for 6 months. The 4 patients with active/progressive disease showed the persistence of disease. This clinical observation may be correlated with in vitro studies, indicating that AML cells are capable to induce NK cell apoptosis in a dose-depend manner. In summery, a two-step enrichment of CD56+ NK cells allows the collection of a suitable number of target cells to be used as adoptive immunotherapy in AML patients. Infusion of NK cells is feasible and safe and adoptively transferred NK cells can be detected after infusion.

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Total ankle arthroplasty (TAA) is still not as satisfactory as total hip and total knee arthroplasty. For the TAA to be considered a valuable alternative to ankle arthrodesis, an effective range of ankle mobility must be recovered. The disappointing clinical results of the current generation of TAA are mostly related to poor understanding of the structures guiding ankle joint mobility. A new design (BOX Ankle) has been developed, uniquely able to restore physiologic ankle mobility and a natural relationship between the implanted components and the retained ligaments. For the first time the shapes of the tibial and talar components in the sagittal plane were designed to be compatible with the demonstrated ligament isometric rotation. This resulted in an unique motion at the replaced ankle where natural sliding as well as rolling motion occurs while at the same time full conformity is maintained between the three components throughout the flexion arc. According to prior research, the design features a spherical convex tibial component, a talar component with radius of curvature in the sagittal plane longer than that of the natural talus, and a fully conforming meniscal component. After computer-based modelling and preliminary observations in several trial implantation in specimens, 126 patients were implanted in the period July 2003 – December 2008. 75 patients with at least 6 months follow-up are here reported. Mean age was 62,6 years (range 22 – 80), mean follow-up 20,2 months. The AOFAS clinical score systems were used to assess patient outcome. Radiographs at maximal dorsiflexion and maximal plantar flexion confirmed the meniscalbearing component moves anteriorly during dorsiflexion and posteriorly during plantarflexion. Frontal and lateral radiographs in the patients, show good alignment of the components, and no signs of radiolucency or loosening. The mean AOFAS score was observed to go from 41 pre-op to 74,6 at 6 month follow-up, with further improvement at the following follow-up. These early results reveal satisfactory clinical scores, with good recovery of range of motion and reduction of pain. Radiographic assessment reveals good osteointegration. All these preliminary results confirm biomechanical studies and the validity of this novel ligamentcompatible prosthesis design. Surely it will be important to re-evaluate these patients later.

The use of scaffolds for Tissue Engineering (TE) is increasing due to their efficacy in helping the body rebuild damaged or diseased tissue. Hydroxyapatite (HA) is the most suitable bioactive ceramic to be used in orthopaedic reconstruction since it replicates the mineral component of the hard tissues, and it has therefore excellent biocompatibility properties. The temporal and spatial control of the tissue regeneration process is the limit to be overcome in order to treat large bone and osteochondral defects. In this thesis we describe the realization of a magnetic scaffolds able to attract and take up growth factors or other bio-agents in vivo via a driving magnetic force. This concept involves the use of magnetic nanoparticles (MNP) functionalized with selected growth factors or stem cells. These functionalized MNP act as shuttles transporting the bio-agents towards and inside the scaffold under the effect of the magnetic field, enhancing the control of tissue regeneration processes. This scaffold can be imagined as a fixed “station” that provides a unique possibility to adjust the scaffold activity to the specific needs of the healing tissue. Synthetic bone graft substitutes, made of collagen or biomineralized collagen (i.e. biomimetic Hydroxyapatite/collagen composites) were used as starting materials for the fabrication of magnetic scaffolds. These materials are routinely used clinically to replace damaged or diseased cartilaginous or bone tissue. Our magnetization technique is based on a dip-coating process consisting in the infilling of biologically inspired porous scaffolds with aqueous biocompatible ferrofluids’ suspensions. In this technique, the specific interconnected porosity of the scaffolds allows the ferrofluids to be drawn inside the structure by capillarity. A subsequent freeze-drying process allows the solvent elimination while keeping very nearly the original shape and porosity of the scaffolds. The remaining magnetic nanoparticles, which are trapped in the structure, lead to the magnetization of the HA/Collagen scaffold. We demonstrate here the possibility to magnetize commercially available scaffolds up to magnetization values that are used in drug delivery processes. The preliminary biocompatibility test showed that the investigated scaffolds provide a suitable micro-environment for cells. The biocompatibility of scaffold facilitates the growth and proliferation of osteogenic cells.

Purpose: Recent knowledge regarding tissue biology highlights a complex regulation of growth factors in reaction to tissue damage. Platelet Rich Plasma (P.R.P.), containing a natural pool of growth factors, can be obtained in a simple and minimally invasive way and be applied to the lesion site. The aim of this study is to explore this novel approach to treat cartilage degenerative lesions of the knee and tendon chronic lesions( patellar tendon, and achilles tendon). In this study we evaluated if the treatment with PRP injections can reduce pain and increase function in cases of patellar tendinosis (Jumper’s Knee), in chronic achilles tendinopathy and in patients with cartilage injuries of the knee. Materials and Methods: 40 patients with cartilage lesion of the knee, 28 male and 12 female with mean age 47 y. (min 18- max 52 years), were treated and prospectively evaluated at a minimum 6 months follow-up; in the same way, 12 patients with achilles tendon lesion (8 male and 4 female) with mean age 44,5 y. (min 32-max 58 years) and 10 patients with “Jumper’s Knee” (8 male and 2 female) with mean age 23,2 y. (min 18-max 37 years), were evaluated at 6 months follow up. The procedure involved 3 multiple injections , performed every two weeks. All patients were clinically evaluated at the end of the treatment and at 6 months follow up. IKDC, SF36, EQ-VAS, scores were used for clinical evaluation and patient satisfaction and functional status were also recorded. Results: Statistical analysis showed a significant improvement in the SF36 questionnaire in all parameters evaluated at the end of the therapy and 6 months follow-up in both group(tendinopathies and chondral lesions), and in the EQ VAS and IKDC score (paired T-test, p<0.0005) from basal evaluation to the end of the therapy, and a further improvement was present at 6 months follow-up. Whereas a higher improvement of the sport activity level was achieved in the “Jumper’s Knee” group. No complications related to the injections or severe adverse events were observed during the treatment and follow up period. Conclusion: PRP inhibits excess inflammation, apoptosis, and metalloproteinase activity. These interactive pathways may result in the restoration of tendon or cartilage, which can with stand loading with work or sports activity, thereby diminishing pain. PRP may also modulate the microvascular environment or alter efferent or afferent neural receptors. The clinical results are encouraging, indicating that PRP injections may have the potential to increase the tendon and cartilage healing capacity in cases with chronic tendinosis and chondropathy of the knee.

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