Tailoring the properties of composite scaffolds with a 3D-Printed lattice core and a bioactive hydrogel shell for tissue engineering.

The optimal performance of scaffolds for tissue engineering relies on a proper combination of their constituent biomaterials and on the design of their structure. In this work, composite scaffolds with a core-shell architecture are realized by grafting a gelatin-chitosan hydrogel onto a 3D-printed polylactic acid (PLA) core, aiming in particular at bone regeneration. This hydrogel was recently found to sustain osteogenic differentiation of mesenchymal stromal cells, leading to new bone tissue formation. Here, the integration with rigid PLA lattice structures provides improved mechanical support and finer control of strength and stiffness. The core is prepared by fused deposition modeling with the specific aim to study several lattice structures and thereby better tune the scaffold mechanical properties. In fact, the core architecture dictates the scaffold strength and stiffness, which are seen to match those of different types of bone tissue. For all lattice types, the hydrogel is found to penetrate throughout the entire core and to present highly interconnected pores for cell colonization. By varying the void volume fraction in the core it is possible to significantly change the bioactive shell content, as well as the mechanical properties, over a wide range of values. Looking for design guidelines, relationships between stiffness/strength and density are here outlined for scaffolds featuring different lattice parameters. Moreover, by acting on the core strut arrangement, scaffolds are reinforced along specific directions, as evaluated under compressive and bending loading conditions.

In vitro evaluation of TAT-OP1 osteogenic properties and prospects for in vivo applications.

So far, osteogenic protein 1 (OP1) is biotechnologically produced and approved for the treatment of human lumbar spine fusion and long bone non-union fractures. When combined with the TAT sequence, it has been demonstrated in vitro to be easily taken up by PC12 neuronal cells and to acquire its biological activity after intracellular refolding. In this study, TAT-OP1 was shown to be a useful strategy to efficiently drive denatured OP1 into mouse MC3T3E1 pre-osteoblasts. The correct in vitro protein refolding was verified by the activation of the BMP cascade, while the osteogenic potential of OP1 was demonstrated by increased expression of alkaline phosphatase, osteonectin and osteocalcin.

Electrochemotherapy for disseminated superficial metastases from malignant melanoma.

BACKGROUND: The aim of the study was to determine predictive factors for effectiveness, toxicity and local disease control in patients with malignant melanoma treated with bleomycin-based electrochemotherapy (ECT). METHODS: Electrochemotherapy was offered to patients with superficially disseminated melanoma metastases unsuitable for resection and unresponsive to chemotherapy. RESULTS: Eighty-five patients were treated with up to six ECT cycles with minimal, mainly dermatological, toxicity. One month after the first ECT, an objective response was observed in 80 patients (94 per cent). After retreatment because of a partial response in 39 patients, a complete response was achieved in 19 patients. Among the 41 (48 per cent) complete responders at first ECT, 19 patients received a second cycle because of new lesions after a median of 6 (range 2-14) months. After a median follow-up of 26 months, six patients experienced local recurrence with a 2-year local progression-free survival rate of 87 per cent. In multivariable analysis, significant predictive factors for response were tumour size (odds ratio (OR) 0·23, 95 per cent confidence interval (c.i.) 0·19 to 0·86; P = 0·003) and number of lesions (OR 0·38, 0·28 to 0·88; P = 0·002). An increasing number of electrode applications (hazard ratio (HR) 2·18, 95 per cent c.i. 1·22 to 3·44; P = 0·041) and ECT cycles (HR 0·46, 0·22 to 0·95; P = 0·005) were predictors of local control. There were no predictors of toxicity. Melanoma thickness and lower limb location of metastases were prognostic for survival. CONCLUSION: The most suitable candidates for ECT were patients with few and small metastases on the lower limb treated with multiple electrode applications and ECT cycles.

Ear melanoma: influence of perichondrium involvement in evaluating surgical strategy.

Adequate treatment of melanomas of the external ear offers unique surgical challenges because of the complex anatomical features of the auricle and the need for proper oncologic aggressiveness, as well as for valid aesthetic results. In this study, we evaluated nine different cases of melanoma of the auricle treated in our Institute between 1994 and 2008. Every enrolled patient underwent surgical excision, nonetheless reconstruction was performed with different surgical techniques. In accordance with histological reports, we could observe the absence of neoplastic cells in perichondral bone and in cartilagenous tissue, providing further assurance concerning the importance of preserving the outer ear in expectation of reconstruction. As a matter of fact, it has been proven that it is possible to reconstruct the auricle while guaranteeing oncologic safety in melanomas with a Breslow thickness >1 mm. Therefore, an excision preserving the cartilagenous tissue, thus allowing an efficient full-thickness cutaneous grafting may guarantee good functional and aesthetic results. In conclusion, the suggested therapeutic management may be considered in <1 mm melanomas without taking further unnecessary oncologic risks.

Masson's disease in hand surgery: a clinicopathologic study of four cases.

Intravascular papillary endothelial hyperplasia (IPEH), also known as Masson's tumour, is an uncommon benign vascular lesion characterized by small multiple, endothelial-lined, papillary structures with hyaline stalks. It appears to be a reactive condition involving excessive proliferation of endothelial cells in normal blood vessels or in vascular malformations, perhaps in response to blood vessel injury or thrombosis. The lesions are small, superficial, reddish-blue nodules, usually in the head, neck or hand. Distinction from pyogenic granuloma and angiosarcoma usually requires pathological examination. Complete surgical excision is the treatment of choice. Four cases affecting the finger are presented together with histological features.

Polymer-coated quartz crystal microbalance chemical sensor for heavy cations in water.

A flow type quartz crystal microbalance (QCM) (bio)chemical sensor was developed for the real time determination of heavy metal ions that is suitable for environmental monitoring. A new process has been developed which enables to obtain surface-modified gold electrodes with high heavy metal ions complexing ability. The sensing performances of the piezoelectric sensor used in a flow-through setup were investigated by monitoring the frequency variation induced by the presence of heavy metal ions, such as copper and lead, as model ions, in aqueous media. X-Ray Reflectivity (XRR) and Atomic Force Microscopy (AFM) were carried out to characterize the unmodified and modified gold surfaces.

Synthesis and pharmacokinetic behaviour of ester derivatives of 4-isobutylphenyl-2-propionic acid (Ibuprofen) with end-hydroxylated poly(N-vinyl pyrrolidinone) and poly(N-acryloyl morpholine) oligomers.

Four derivatives of 4-isobutylphenyl-2-propionic acid (Ibuprofen), in which the drug was bound by ester linkages to poly(ethylene glycols) (PEG 2000-I), monomethoxy poly(ethylene glycols) (PEG 1900-I), poly(N-vinyl pyrrolidinone) (PVP-I) and poly(N-acryloyl morpholine) (PACM-I), all having approximatively the same number average molecular weight (Mn congruent equal to 2000), were prepared and tested for their pharmacokinetic properties after oral administration. It was found that the two end-hydroxylated amphiphilic oligomers of polyvinylic structure, PACM and PVP, whose physico-chemical properties are comparable to those of PEGs especially as regards solvent affinity, have in principle a similar potential as promoieties for preparing oligomeric prodrugs.

Degradation behaviour of ionic stepwise polyaddition polymers of medical interest.

The aim of this presentation is to review some of our recent work mostly on poly(amidoamine)s (PAAs) and some other families of polymers structurally related to PAAs of medical interest. PAAs are obtained by stepwise polyaddition of primary monoamines, or bis secondary amines, to bisacrylamides. There are several other ter-amino polymers structurally related to PAAs, such poly(amido phosphine)s (PAPs), poly(ester-amine)s (PEAs), poly(ketone-amine)s (PKAs), poly(amidothioeteramine)s (PATAs) poly(esterthioether amine)s (PTEAs), and poly(sulphone thioetheramine)s (PSTAs). Most of the PAAs exhibit heparin complexing ability. PAAs are also being considered as soluble carriers for delivering anti-cancer drugs. Some of these polymers have been studied as antimicrobial agents. PAAs with different structures degrade at different rates under physiological conditions. The degradation rate is also strongly influenced by pH. The quaternarized PATAs and PTEAs are reasonably stable over a period of some days, but ultimately degrade to oligomeric products, while the quaternized PAAs do rapidly degrade.

Recent results on functional polymers and macromonomers of interest as biomaterials or for biomaterial modification.

Different families of functionalized polymers with potential as biomaterials, or for biomaterial modification, have been investigated. In particular, degradation studies have been performed on poly(amidoamines), a family of polymers obtained by polyaddition of amines to bisacrylamides, and endowed with heparin-complexing ability. Some new poly(amidoamines) with more resistance towards hydrolytic degradation than traditional ones have been discovered. Other ter-amino polymers deriving from the polyaddition of ter-amino functionalized bis-thiols to bis-acrylic esters, or other activated unsaturated compounds, have been studied. Their quaternarization products have been proven, in a parallel work, to act as powerful antimicrobial agents. By performing in situ the polyaddition reaction, semi-interpenetrated networks based on silicone rubber and the same polymers have been prepared. Finally, end-functionalized amphiphilic oligomers have been prepared by radical polymerization techniques, and their use for enzyme modification considered.

Pharmacokinetic results on naproxen prodrugs based on poly(ethyleneglycol)s.

Five prodrugs of S(+)-2-(6-methoxy-2-naphthyl)propionic acid (naproxen), in which the drug was bound by ester linkages to diethyleneglycol (I), triethyleneglycol (II), octanediol (III), butyl-triethyleneglycol (IV), and butyl-tetraethyleneglycol (V), respectively, were prepared and tested for their pharmacokinetic properties after oral administration. It was found that bioavailabilities decreased in the order, and in all cases were lower than that of the free drug.

Preparation and properties of monomethoxy poly(ethylene glycol) doxorubicin conjugates linked by an amino acid or a peptide as spacer.

Polymeric doxorubicin prodrugs were prepared linking monomethoxy poly(ethylene glycol), 5000 D molecular weight, to the doxorubicin amino group, using an amino acid or a peptide as a spacer arm. As spacers glycine, L-phenylalanine, L-tryptophan and glycil-L-valil-L-phenylalanine were used. The conjugates showed enhanced stability to alkaline degradation compared to the free doxorubicin. Towards Ehrlich solid tumor in mice the glycin spaced derivative was devoid of activity, whereas the phenylalanine and tryptophan derivatives were 20% and 16% active and the tripeptide one 50% active with respect to free doxorubicin. On the other hand the derivatization was accompanied by a great decrease of toxicity in mice with respect to the free drug. Doxorubicin was not released from conjugates by chymotrypsin incubation or in plasma.

Accurate evaluation method of the polymer content in monomethoxy(polyethylene glycol) modified proteins based on amino acid analysis.

To overcome the uncertainty of the colorimetric or fluorimetric method so far employed for the evaluation of monomethoxy(polyethylene glycol) (MPEG) covalently bound to protein, a direct method based on amino acid analysis is proposed. The method exploits the use of MPEG, which was bounded with the unnatural amino acid norleucine (MPEG-Nle). MPEG-Nle was activated at its carboxylic group to succinimidyl ester for the binding to the amino groups of protein. After acid hydrolysis, the amino acid content is evaluated by conventional amino acid analyzer or by reverse-phase HPLC as phenylthiocarbamyl derivative. The number of bound MPEG chains is calculated from the amino acid composition, since one norleucine residue is released from each bound polymer chain. The method was verified with several proteins in comparison with colorimetric ones, also in the case of proteins that contain chromophores in the visible range, such cytocrome C. It was observed that in most of the cases, the colorimetric methods give an overestimation of the degree of protein modification.

Surface modification of enzymes for therapeutic use: monomethoxypoly (ethylene glycol) derivatization of ribonuclease.

Bovine pancreatic ribonuclease A (RNase) was modified at various extent at the lysine residues by monomethoxypoly(ethylene glycol) (MPEG) activated as active ester. For pharmacokinetic experiments a radioactive adduct was also prepared with tritiated amino acid as spacer between polymer and protein. The modification reduced only slightly the RNase catalytic activity and Km towards the substrate cytidine-2',3'-cyclic monophosphate. On the other hand extensively modified MPEG-RNase samples, showed significant decrease in activity towards ribonucleic acid. The polymer modification did not change the pH activity profile, increased the stability to proteolytic digestion, while the behaviour towards denaturants and heat was not modified. The native and MPEG-RNase administered IV, IM and SC to rats, showed impressive differences in pharmacokinetics: the half-life of the modified enzyme, evaluated in blood by radioactivity, was increased of 40-50 folds with respect to the native form.

Anti-inflammatory activity of monomethoxypolyethylene glycol superoxide dismutase on adjuvant arthritis in rats.

In previous studies we observed an enhanced anti-inflammatory activity of MPEG-SOD derivatives in acute inflammation in rat. To assess the activity in chronic inflammation we tested the compound with longer half-life (MPEG-SOD 18) in complete adjuvant arthritis in rat. According to the prophylactic schedule of treatment (i.m. administration at alternative days of 10 mg/kg from day 3 to day 21), the MPEG-SOD derivative reduced arthritic lesions in a significant way (P less than 0.01 at 14th, 21st, 28th day). Indomethacin, administered i.m. daily at the dose of 1.5 mg/kg according to the same schedule, significantly inhibited adjuvant arthritis each time it was considered (% of inhibition are 66.5% at 14th day, 58.3% at 21st day and 50.8% at 28th day). Native SOD and inactivated enzyme, administered from day 3 to day 21 did not show any anti-inflammatory properties. According to the therapeutic schedule of treatment (from day 14 to day 28), neither MPEG-SOD nor native SOD showed antiarthritic activity.

Enzyme modification by MPEG with an amino acid or peptide as spacer arms.

A method for the modification of enzymes by MPEG carrying an amino acid or peptide as a spacer arm is described and tested with aliphatic or aromatic side chains amino acids. The procedure involves MPEG activation by p-nitrophenylchloroformate for the amino acid or peptide coupling that is in turn activated for the protein binding. The advantage of the method resides in the possibility to introduce proper reporter groups between the polymer and the protein as norleucine for a direct evaluation of the bound polymer chains, tryptophan for structural studies of the polymer-protein adduct, and radioactive amino acid for pharmacokinetic investigations. The method was positively tested with arginase, ribonuclease, and superoxide dismutase as enzymes of therapeutic value.

Critical main-chain length for conformational conversion from 3(10)-helix to alpha-helix in polypeptides.

To assess the minimal peptide length required for the stabilization of the alpha-helix relative to the 3(10)-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula-(Aib-L-Ala)n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3(10)-helical with four 1----4 intramolecular N-H . . . O = C H-bonds. On the other hand, the octapeptide molecules are essentially alpha-helical with four 1----5 H-bonds; however, the helix is elongated at the N-terminus, with two 1----4 H-bonds, giving these molecules a mixed alpha/3(10)-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3(10)----alpha-helix conversion in the crystal state induced by peptide backbone lengthening only.

A direct assay for evaluation of polyethylene glycol in enzyme adducts used as drugs or biocatalysts.

A specific and direct method for the evaluation of monomethoxypolyethylene glycol (MPEG) in enzyme adducts has been developed. The method is based on extensive modification by trinitrobenzensulfonic acid (TNBS) of the MPEG-enzyme, to form an acid-stable TNBS adduct with the available amino groups of lysine and alpha-amino acid. The MPEG-modified trinitrophenylated enzyme was hydrolyzed in 6N HCl and amino acid composition evaluated by a standard AA analyzer in comparison with the analysis of a MPEG enzyme sample. The number of bound MPEG polymers was calculated from the difference in amino acid composition of the two samples.

Evaluation of proteolytic enzymes in effervescent tablets.

A procedure for the evaluation of proteolytic enzymes in effervescent tablets is reported. A proper chromatographic step followed by enzymatic activity assay allows to overcome the interferences due to the high salt content of such formulations as well as to chromophores eventually present in the tablet components.