Magnetic poly(ε-caprolactone)/iron-doped hydroxyapatite nanocomposite substrates for advanced bone tissue engineering.

In biomedicine, magnetic nanoparticles provide some attractive possibilities because they possess peculiar physical properties that permit their use in a wide range of applications. The concept of magnetic guidance basically spans from drug delivery and hyperthermia treatment of tumours, to tissue engineering, such as magneto-mechanical stimulation/activation of cell constructs and mechanosensitive ion channels, magnetic cell-seeding procedures, and controlled cell proliferation and differentiation. Accordingly, the aim of this study was to develop fully biodegradable and magnetic nanocomposite substrates for bone tissue engineering by embedding iron-doped hydroxyapatite (FeHA) nanoparticles in a poly(ε-caprolactone) (PCL) matrix. X-ray diffraction analyses enabled the demonstration that the phase composition and crystallinity of the magnetic FeHA were not affected by the process used to develop the nanocomposite substrates. The mechanical characterization performed through small punch tests has evidenced that inclusion of 10 per cent by weight of FeHA would represent an effective reinforcement. The inclusion of nanoparticles also improves the hydrophilicity of the substrates as evidenced by the lower values of water contact angle in comparison with those of neat PCL. The results from magnetic measurements confirmed the superparamagnetic character of the nanocomposite substrates, indicated by a very low coercive field, a saturation magnetization strictly proportional to the FeHA content and a strong history dependence in temperature sweeps. Regarding the biological performances, confocal laser scanning microscopy and AlamarBlue assay have provided qualitative and quantitative information on human mesenchymal stem cell adhesion and viability/proliferation, respectively, whereas the obtained ALP/DNA values have shown the ability of the nanocomposite substrates to support osteogenic differentiation.

A conceptually new type of bio-hybrid scaffold for bone regeneration.

Magnetic bio-hybrid porous scaffolds have been synthesized, nucleating nano-apatite in situ on self-assembling collagen, in the presence of magnetite nano-particles. The magnetic phase acted as a sort of cross-linking agent for the collagen, inducing a chemico-physical-mechanical stabilization of the material and allowing us to control the porosity network of the scaffold. Gradients of bio-mineralization and magnetization were also developed for osteochondral application. The good potentiality of the material as a biomedical device, able to offer assistance to bone regeneration through scaffold reloading with specific factors guided by an external magnetic field, has been preliminarily investigated. Up to now the proof of this concept has been realized through in vitro assessments.

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells.

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

Effects of isolated limb perfusion with tumor necrosis factor-alpha on circulating levels of proinflammatory cytokines.

Hyperthermic isolated limb perfusion (ILP) with tumor necrosis factor-a (TNFalpha) and cytotoxic drugs is currently used for treatment of melanoma and sarcoma of the limbs. Tumor necrosis factor-alpha is involved in the systemic inflammatory response syndrome as a result of activation of inflammatory cells and production of bioactive substances. The goal of this study was to determine the circulating levels of proinflammatory cytokines and soluble adhesion molecules in 19 patients with limb melanoma or sarcoma undergoing ILP with (n = 9) or without TNFalpha (n = 10). The results obtained demonstrated that ILP with TNFalpha was responsible for a leakage of TNFalpha in the systemic circulation, followed by a rise in interleukin (IL)-6 and IL-8 levels within I h. Elevated soluble (s)P-selectin levels were found 1-3 h after ILP. Plasma sE-selectin peaked 6-9 h after ILP, and soluble vascular cell adhesion molecule (sVCAM) levels reached a maximum after 24 h. Significant correlations were observed among these variables, confirming the interdependence of all changes observed. On the other hand, ILP with cytotoxic drugs alone induced only a modest release of TNFalpha, which was not followed by an immediate rise in IL-6 and IL-8. Four of the 9 patients undergoing ILP with TNF had severe systemic toxicity. No association was found between systemic TNF levels and the clinical outcome, whereas elevated TNF perfusion levels as well as systemic IL-6 and IL-8 levels were constantly elevated in patients with severe toxicity. These results are suggestive of an important role of TNFalpha levels in the perfusion system (more than leakage of perfusate) in causing postoperative toxicity, although other ILP-related factors should not be excluded.

Treatment of metastatic bone pain with tin-117m Stannic diethylenetriaminepentaacetic acid: a phase I/II clinical study.

The physical characteristics of Sn-117m combined with the biodistribution of the compound tin-117m (Stannic, 4+) diethylenetriaminepentaacetic acid (Sn-117m DTPA) suggest that it should be an excellent agent for the palliation of pain from bony metastases. Prior work has established the dosimetry and the safety for the material in human beings. The presence of low-energy conversion electrons should result in the relative sparing of the bone marrow while delivering a high radiation dose to sites of bony metastatic disease. Forty-seven patients with painful bone metastases from various malignancies were treated with Sn-117m DTPA. The patients were assigned to five different dose levels ranging from 2.64 to 10.58 MBq (71-286 microCi) per kg of body weight. Follow-up included review of pain diaries, performance scores, analgesic requirements, blood chemistries, and hematological assessment. Three patients received a second treatment. There was an overall response rate for relief of pain of 75% (range, 60-83%) in the 40 treatments that could be evaluated. No correlation was apparent in this limited series between response rate and the five dose levels used. The relief was complete in 12 patients (30%). The time to onset of pain relief was 19 +/- 15 days with doses < or = 5.29 MBq/kg and 5 +/- 3 days with doses > or = 6.61 MBq/kg. Myelotoxicity was minimal, with only one patient having a marginal grade 3 WBC toxicity. On the basis of our data, Sn-117m DTPA should be an effective and safe radiopharmaceutical for palliation of painful bony metastases. A large-scale trial is warranted to evaluate it in comparison to other similar agents.

Tin-117m(4+)-DTPA for palliation of pain from osseous metastases: a pilot study.

UNLABELLED: The physical and biological attributes of 117mSn(4+)-DTPA indicate that it should be an effective agent for palliative therapy of painful bony metastatic disease. The aim of this study was to evaluate whether or not this agent could effectively reduce pain while sparing the hemopoietic marrow from adverse effects. METHODS: Fifteen patients (10 males and 5 females) with painful bony metastases from various primary cancers were included in the study. Seven patients received 1.22 to 3.11 MBq/kg of 117mSn intravenously (Group 1) and eight patients received 4.85 to 5.77 MBq/kg (Group 2). All but one were treated as outpatients and followed for a minimum of 2 mo. RESULTS: In the first group, pain relief was non-assessable in four patients because of death or additional treatment of soft-tissue disease by another modality. One patient had no relief of pain, one had complete relief of pain and one had transient relief of pain. No myelotoxicity was observed. For Group 2, three patients achieved complete relief of pain, two good relief, two partial relief and one began to experience pain relief when he suffered a pathological fracture 2 mo post-treatment. None of these patients had myelotoxicity. CONCLUSION: Tin-117m(4+)-DTPA can reduce pain from metastatic disease to bone without inducing adverse reactions related to bone marrow. Further studies are needed to assess tolerance levels for the bone marrow and to evaluate response rates and duration of effect.