Bioactive injectable hydrogels for on demand molecule/cell delivery and for tissue regeneration in the central nervous system.

Currently there are no potential curative therapies that can improve the central nervous system (CNS) regeneration after traumatic injuries or diseases. Indeed, the regeneration of CNS is greatly impaired by limited drug penetration across the blood brain barrier (BBB), poor drug targeting, deficient progenitor neural cells and limited proliferation of mature neural cells. To overcome these limitations, bioengineered injectable hydrogels in combination with drug and cell therapy have been proposed to mimic the complexity of the CNS microenvironment and architecture. Additionally, to enhance relevant CNS regeneration, proper biophysical and biochemical cues are needed. Recently, great efforts have been devoted to tailor stimuli-responsive hydrogels as novel carrier systems which are able to guide neural tissue regeneration. This review provides an extensive overview on the most promising injectable hydrogels for neural tissue engineering. A special emphasis is made to highlight the ability of these hydrogels to deliver bioactive compounds/cells upon the exposure to internal and external stimuli. Bioactive injectable hydrogels have a broad application in central nervous system's (CNS) regeneration. This review gives an overview of the latest pioneering approaches in CNS recovery using stimuli-responsive hydrogels for several neurodegenerative disorders. STATEMENT OF SIGNIFICANCE: This review summarizes the latest innovations on bioactive injectable hydrogels, focusing on tailoring internal/external stimuli-responsive hydrogels for the new injectable systems design, able to guide neural tissue response. The purpose is to highlight the advantages and the limitations of thermo-responsive, photo responsive, magnetic responsive, electric responsive, ultrasound responsive and enzymes-triggered injectable hydrogels in developing customizable neurotherapies. We believe that this comprehensive review will help in identifying the strengths and gaps in the existing literature and to further support the use of injectable hydrogels in stimulating CNS regeneration.

New hydroxyapatite nanophases with enhanced osteogenic and anti-bacterial activity.

This work describes the synthesis and characterization of new apatite phases co-doped with gallium, magnesium and carbonate, exhibiting osteogenic and antibacterial ability. The apatites are synthesized at low temperature to retain nanocrystallinity and controlled doping with the various bioactive foreign ions, as assessed by physico-chemical and crystallographic analyses, reporting the achievement of single phases with reduced crystal ordering. The analysis of single and multi-doped apatites reports to different mechanisms acting in the incorporation of gallium and magnesium ions in the apatite structure. The release of bioactive ions is correlated to the behavior of human mesenchymal stem cells and of different bacterial strands, selected among the most frequently affecting surgical procedures. Enhanced osteogenic and antibacterial ability is assessed in multi-doped apatites, thus suggesting potential future applications as new smart biomaterials integrating a significant boosting of bone regeneration with adequate protection against bacteria. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 106A: 521-530, 2018.

Surface morphology, tribological properties and in vitro biocompatibility of nanostructured zirconia thin films.

Deposition of nanostructured and low-wear zirconia (ZrO2) thin films on the metallic component of a total joint implant is envisaged to reduce wear of the soft ultra-high molecular weight polyethylene (UHMWPE) counterpart. In this work, morphological surface features, wear resistance and in vitro-biocompatibility of zirconia thin films deposited by the novel Pulsed Plasma Deposition (PPD) method have been investigated. Film thickness, roughness and wettability were found to be strongly dependent on deposition gas pressure. Interestingly, wear rate of UHMWPE disks coupled to zirconia-coated titanium spheres was only poorly correlated to the contact angle values, while film roughness and thickness seemed not to affect it. Furthermore, wear of UHMWPE, when coupled with zirconia coated-titanium spheres, significantly decreased with respect to uncoated spheres under dry or NaCl-lubricated conditions; besides, when using bovine serum, similar results were obtained for coated and uncoated spheres. Finally, suitable mesenchymal stem and osteoblast cells adhesion, proliferation and viability were observed, suggesting good biocompatibility of the nanostructured zirconia films. Taken together, the results shown in this work indicate that zirconia thin films deposited by the PPD method deserve further investigations as low-wear materials for biomedical applications such as total joint replacement.

A comparison between micro-CT and histology for the evaluation of cortical bone: effect of polymethylmethacrylate embedding on structural parameters.

Cortical bone microstructure is an important parameter in the evaluation of bone strength. The aim of this study was to validate the characterization of human cortical bone microarchitecture using microcomputed tomography. In order to do this, microcomputed tomography structural measurements were compared with those obtained through histological examination (the gold standard). Moreover, to calculate structural parameters, microcomputed tomography images have to be binarized with the separation between bone and nonbone structures throughout a global thresholding. As the effect of the surrounding medium on the threshold value is not clear, an easy procedure to find the global uniform threshold for a given acquisition condition is applied. This work also compared the structural parameters of microcomputed tomography cortical sample scan in air or embedded in polymethylmethacrylate; histology was used as a reference. For each acquisition condition, a fixed threshold value was found and was applied on the corresponding microcomputed tomography image for the parameters assessment. Twenty cortical bone samples were collected from human femur and tibia diaphyses. All samples were microcomputed tomography scanned in air, embedded in polymethylmethacrylate, rescanned by microcomputed tomography, examined by histology and finally compared. A good correspondence between the microcomputed tomography images and the histological sections was found. Paired comparisons in cortical porosity, Haversian canal diameter and Haversian canal separation between histological sections and microcomputed tomography cross sections, first in air and then embedded in PolyMethylMethAcrylate, were made: no significant differences were found. None of the comparisons showed significant differences for cortical porosity, Haversian canal diameter and Haversian separation over a three-dimensional volume of interest, between microcomputed tomography scans in air and with samples embedded in PolyMethylMethAcrylate. The very good correlation between bone structural measures obtained from microcomputed tomography datasets and from two-dimensional histological sections confirms that microcomputed tomography may be an efficient tool for the characterization of cortical bone microstructure. Moreover, when the corresponding threshold value for each condition is used, structural parameters determined by microcomputed tomography are not affected by the surrounding medium (PolyMethylMethAcrylate).

18F-FDG positron autoradiography with a particle counting silicon pixel detector.

We report on tests of a room-temperature particle counting silicon pixel detector of the Medipix2 series as the detector unit of a positron autoradiography (AR) system, for samples labelled with (18)F-FDG radiopharmaceutical used in PET studies. The silicon detector (1.98 cm(2) sensitive area, 300 microm thick) has high intrinsic resolution (55 microm pitch) and works by counting all hits in a pixel above a certain energy threshold. The present work extends the detector characterization with (18)F-FDG of a previous paper. We analysed the system's linearity, dynamic range, sensitivity, background count rate, noise, and its imaging performance on biological samples. Tests have been performed in the laboratory with (18)F-FDG drops (37-37 000 Bq initial activity) and ex vivo in a rat injected with 88.8 MBq of (18)F-FDG. Particles interacting in the detector volume produced a hit in a cluster of pixels whose mean size was 4.3 pixels/event at 11 keV threshold and 2.2 pixels/event at 37 keV threshold. Results show a sensitivity for beta(+) of 0.377 cps Bq(-1), a dynamic range of at least five orders of magnitude and a lower detection limit of 0.0015 Bq mm(-2). Real-time (18)F-FDG positron AR images have been obtained in 500-1000 s exposure time of thin (10-20 microm) slices of a rat brain and compared with 20 h film autoradiography of adjacent slices. The analysis of the image contrast and signal-to-noise ratio in a rat brain slice indicated that Poisson noise-limited imaging can be approached in short (e.g. 100 s) exposures, with approximately 100 Bq slice activity, and that the silicon pixel detector produced a higher image quality than film-based AR.

[The diabetic foot seen by the surgeon. Personal experience]. / Il piede diabetico visto dal chirurgo. Esperienza personale.

BACKGROUND: The aim of this study is to evaluate a major amputation risk criterion in diabetic patients with trophic lesions of the foot. The records of a series of 100 consecutive patients (65 males and 35 females) with diabetic foot ulcer treated in our surgical facilities between January 1992 and December 1997, in collaboration with diabetologists and podiatrists, have been reviewed retrospectively. METHODS: In 26 cases the ulcer involved both limbs and, therefore, the feet observed in this study have been 126. Accurate diagnosis of the underlying cause was the first step and in cases with a poor blood supply (69 limbs; 55%) unresponsive to medical therapy (44 limbs) vascular reconstruction (37 limbs), spinal cord stimulator (SCS) implantation (3 limbs) or major amputation (4 limbs) were performed. According to Wagner grading there were 42 grade 2 ulcers (33%), 38 grade 3 (30%), 43 grade 4 (34%) ad 3 grade 5 (3%). RESULTS: One patient died postoperatively after SCS implantation. All but 4 neuropathic ulcers (53 limbs) healed in a mean time (+/- SD) of 5.2 +/- 3.8 months and all but 10 vascular ulcers (59 limbs) healed in a mean time of 6.3 +/- 4.1 months. Of the latter group in 4 cases the patient died before ulcer healing while in 6 cases (8.7%) a major amputation was performed (in 2 cases after vascular reconstruction procedures). Minor amputations of the forefoot have been performed in 23 instances (33%) of vascular ulcer and in 10 cases (17%) of neuropathic ulcer. CONCLUSIONS: Since ischemia is the main risk factor for amputation, it is suggested that a particular effort should be made in improving the vascular diagnostic, both clinical and strumental, capabilities of our diabetologists and podiatrists in order to detect the vascular insufficiency in earlier stages.

Ocular hypertonia and crystalline lens opacities in healthcare workers exposed to ionising radiation.

Some activities performed by healthcare workers may still involve total or partial exposure to ionising radiation exceeding the limit values. In addition to the appearance of crystalline lens opacities which may lead to rays-cataract, recent studies have indicated possible induction of ocular hypertonia in occupationally exposed subjects. The aim of this study was to establish the actual prevalence of ocular hypertonia and crystalline lens opacities in a group of healthcare workers exposed to ionising radiation. The collected data failed to show significant risk of ocular hypertonia and suggested that crystalline lens opacity was not an important indicator of exposure. Notwithstanding, preventive and periodic (every 5 years) ophthalmologic control may prove helpful for medicolegal purposes. Namely, such control would record congenital crystalline lens opacities in many individuals and would thus rule out unjustified claims of occupational disease due to exposure to ionising radiation. Additionally, ophthalmologic control should focus on different and probably more important ocular risks for the radiologists such as the ocular fatigue resulting from a prolonged use of a video display terminal or other diagnostic screens or electrodiaphanoscopes.

Early abnormalities in left ventricular diastolic function of sodium-sensitive hypertensive patients.

The aim of this study was to evaluate whether salt-sensitivity in essential hypertension produces a significant comparative difference in diastolic function and ventricular mass when compared with sodium-resistance. Recent epidemiological data have demonstrated a positive correlation between sodium intake and arterial pressure. Furthermore, a positive correlation has been detected between sodium intake and left ventricular hypertrophy (LVH) independently of arterial pressure. Thirty-one patients who had never been treated before for uncomplicated hypertension were studied. Each subject received a 30 mmol/per day sodium diet for 14 days, supplemented with a further 190 mmol of sodium in the first study week (220 mmol for the first 7 days and 30 mmol for the second 7 days). Throughout the study compliance was assessed by measuring daily urinary sodium excretion. Sodium sensitivity of blood pressure was defined as the difference (5% or more) between blood pressure at the end of the low and high sodium intake periods. On this basis 16 patients were defined as salt-sensitive (SS) and 15 patients as salt-resistant (SR). The two groups were homogeneous for age, sex and race. Baseline mean arterial pressure (MAP) was comparable between SS (108 +/- 1.8 mm Hg) and SR (107 +/- 2.1 mm Hg, P = NS). Each patient was submitted to M-MODE and two-dimensional echocardiogram studies in order to estimate left ventricular mass using the Penn conventional formula and parameters of left ventricular diastolic function. The left ventricular mass measurement showed higher values in the SS group although this did not reach statistical significance (118.4 +/- 4.4 vs 112.0 +/- 4.2 gr/mq, P = NS). Both interventricular septal and posterior wall thickness did not demonstrate significant differences between the two groups. The salt-sensitive group showed impaired left ventricular diastolic function; in particular, the first diastolic peak representing the early maximum of diastolic filling velocity (E) was lower in SS subjects than in SR subjects (71.6 +/- 2.9 vs83.1 +/- 3.3 cm/sec, P < 0.02). No significant difference was detected in the second peak representing the atrial maximum of filling velocity (A) (69.0 +/- 2.3 vs 66.0 +/- 2.0 cm/sec, P = NS). As a consequence the E/A ratio was significantly different (0.73 +/- 0.2 in the SR vs 1.2 +/- 0.04 in the SS group, P < 0.05). Moreover, the peak E integral was lower in SS than in SR subjects (8. 7 +/- 0.6 vs11.2 +/- 0.5 cm, P < 0.005; no difference for the peak A integral (6.0 +/- 0.3 vs 5.7 +/- 0.4 cm, P = NS). The E peak deceleration time was significantly reduced in the SS group (400.3 +/- 13.5 vs 500.9 +/- 12.8 cm/sec, P < 0.001). No significant difference was found for the isovolumetric relaxation time (IVRT) (95.7 +/- 4.3 vs 92.2 +/- 4.0, P = NS). Our data show an impaired diastolic function expressed by a reduced early diastolic filling velocity (peak E) and a significantly abnormal E/A ratio in SS in comparison with SR subjects. Therefore abnormalities in diastolic function are detectable earlier in SS hypertensive subjects than in SR irrespective of actual MAP.

Antiangiogenic, antitumoural and antimetastatic effects of two distamycin A derivatives with anti-HIV-1 Tat activity in a Kaposi's sarcoma-like murine model.

The antiangiogenic, antitumoural and antimetastatic effects of two novel sulphonic derivatives of distamycin A, PNU145156E and PNU153429, were studied in a Kaposi's sarcoma-like tumour model obtained by injecting nude mice with cells releasing extracellular HIV-Tat protein, derived from a tumour which developed in a BK virus/tat transgenic mouse. Both PNU145156E and PNU153429 were administered intraperitoneally every fourth day for three weeks at doses of 100 or 50 mg/kg of body weight respectively, starting one day after injecting the tumour cells. Both drugs delayed tumour growth in nude mice, preventing neovascularization induced by the Tat protein. PNU153429 also significantly reduced the number and size of spontaneous tumour metastases. Both effects on tumour growth and metastases were augmented by treating simultaneously nude mice with 7.5 mg/kg of body weight of minocycline given per os daily for four weeks starting four days after injecting the tumour cells. Neither acute nor chronic toxic side-effects were observed during the life span of treated nude mice. Due to their antiangiogenic and anti-Tat effects, these drugs are promising for the treatment of Kaposi's sarcoma in AIDS patients.

Autosomal recessive hereditary motor and sensory neuropathy with focally folded myelin sheaths: clinical, electrophysiologic, and genetic aspects of a large family.

We describe 10 patients from a large family with early onset motor and sensory neuropathy. Six were still living at the time of the study. In all cases, early motor milestones had been achieved. Mean age at onset of symptoms was 34 months; these included progressive distal and proximal muscle weakness of lower limbs. Pes equinovarus developed in all patients during childhood. Slight facial weakness was present in four patients, and one of them also had bilateral facial synkinesia. Intellectual function was normal in all cases. There was no evidence of thickened peripheral nerves. All three adult patients (mean age, 27 years) were seriously handicapped and wheelchair-bound. Death occurred in the fourth to fifth decade of life and the duration of the illness varied from 27 to 39 years. Motor nerve conduction velocities ranged from 15 to 17 m/sec in the upper limbs of the youngest patients, and were undetectable in the adult patients. Sensitive action potentials were almost always absent. In all patients, auditory evoked potentials showed abnormally delayed interpeak I-III latencies. The most prominent pathologic finding was a highly unusual myelin abnormality consisting of irregular redundant loops and folding of the myelin sheath. The genealogic study gave strong evidence of autosomal-recessive inheritance. The molecular analysis failed to demonstrate either duplication in the chromosome 17p11.2-12, point mutations in the four exons of the PMP-22 (17p11.2) and the six exons of the Po (1q21-q25) genes, or linkage to chromosome 8q13-21.1.

Cumulative trauma disorders of the upper limbs in workers on an agricultural farm.

The work associated with repetitive efforts and inadequate resting periods, strong physical exertion, awkward postures or static positioning exposes workers to the risk of cumulative trauma disorders of the upper limbs. These risk factors are present in many agricultural activities. A study was carried out among workers on an agricultural farm. The workers' histories were taken and they were given periodical medical check-ups. The presence of upper limb disorders was shown in a group of workers. A sample of 42 people was selected for the study by means of specific tests: electromyography, ultrasonography and laser-doppler flowmetry. The tests showed a high incidence of carpal tunnel syndrome and microcirculation disorders. The study confirmed that electromyography, ultrasonography and/or laser-doppler flowmetry are highly useful tools for identifying cumulative trauma disorders.

Growth arrest and suppression of tumorigenicity of bladder-carcinoma cell lines induced by the P16/CDKN2 (p16INK4A, MTS1) gene and other loci on human chromosome 9.

Wild-type P16/CDKN2 (p16INK4A, MTS1) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder-carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild-type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over-expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2-gene expression is modulated by the physiological control of chromosomal regulatory sequence. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder-carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32-33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor-suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder-carcinoma cells when it is over-expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder-cancer progression.

Suprapubic cystotomy as treatment for severe hemorrhagic cystitis after bone marrow transplantation.

We analyzed the success of suprapubic cystotomy in patients with severe hemorrhagic cystitis after bone marrow transplantation. Seventy-three out of 963 patients developed severe hemorrhagic cystitis which resulted in urinary tract obstruction after high-dose cytoreductive therapy. Eleven patients (15%) failed medical treatment and required emergency suprapubic cystotomy. Three of these patients died of other complications prior to resolution of HC. Of the remaining 8 patients who underwent surgery, 4 are alive. The mortality rate was significantly higher in patients who required surgery than in those who responded to medical therapy. Patients whose HC required surgery also had a greater transfusion requirement than those who responded to medical therapy. We conclude that surgical treatment of severe HC should be undertaken only after failure of medical therapy.

Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease.

Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.

Transfer of human chromosome 3 to an ovarian carcinoma cell line identifies three regions on 3p involved in ovarian cancer.

The molecular pathogenesis of ovarian carcinoma involves altered expression of growth factors, activation of oncogenes and loss of tumor suppressor genes. Loss of heterozygosity on chromosomes 3p, 6q, 11p, 17 and 18q was reported as a significant alteration in ovarian cancer. However, no functional proof has been provided of tumor suppressor activity located in these chromosomal regions. We therefore introduced normal human chromosomes 3 and 11 into an ovarian carcinoma cell line by microcell mediated chromosome transfer. Transfer of chromosome 3 induced senescence and growth arrest as well as suppression of tumorigenicity. Tumors induced by chromosome 3 monochromosomic hybrids consistently lost three small regions on 3p, two of which located in 3p23-24.2 and one located in 3p21.1-21.2, suggesting that these chromosomal regions are important for suppression of tumorigenicity of ovarian carcinoma cells. Transfer of chromosome 11 reduced the in vitro growth properties of ovarian cancer cells but did not significantly affect tumorigenicity. These results provide functional evidence for chromosome 3 tumor suppressor activity in ovarian cancer and define the chromosomal regions on 3p involved in the pathogenesis of this tumor. This experimental system, based on functional effects, may be useful for further delimitation and isolation of critical regions on 3p involved in tumor suppression.

Absence of APP713 mutation in Italian and Russian families with schizophrenia.

A recent study has shown a mutation at codon 713 of the amyloid precursor protein (APP) gene in a schizophrenic patient. We have analyzed the MaeIII restriction site caused by that mutation in Italian and Russian families with schizophrenia. No mutations were observed suggesting that the APP713 mutation is unlikely to be linked to the pathogenesis of such a psychiatric disorder.

A new Italian pedigree with early-onset Alzheimer's disease.

We have recently discovered in Torino (Italy) a new pedigree with early-onset Alzheimer's disease. The index patient is a woman who, at the age of 43 years, showed progressive memory impairment and ideomotor apraxia. Several relatives of the patient have had a history of dementia. The ancestors of the patient were from Calabria (southern Italy) and members of the family emigrated to the north of Italy, to France, and to the United States. Up to now, the new kindred comprises 1950 members, distributed in eight generations. Thirty members affected with Alzheimer's disease have been identified. Neuropathologic confirmation of antemortem clinically diagnosed Alzheimer's disease has been achieved for one patient. The pedigree is consistent with autosomal dominant inheritance. The clinical course of the disease is fairly uniform: the first symptom is memory loss, beginning around age 40 years. Psychiatric symptoms like hallucinations and delusions follow. At a later stage of the disease, several patients developed myoclonus and generalized epileptic seizures and eventually died with profound dementia. The "Torino family" shows several genealogic and clinical similarities with other large multigenerational familial Alzheimer's disease pedigrees originating from the Calabria region.

Analysis of the c-FOS gene on chromosome 14 and the promoter of the amyloid precursor protein gene in familial Alzheimer's disease.

The c-FOS gene product, a putative transacting transcriptional regulator of the amyloid precursor protein (APP) gene, is a candidate locus for the familial Alzheimer's disease (FAD) mutation on chromosome 14 (FAD14). In light of this functional relationship, we investigated the nucleotide sequence and segregation of c-FOS and the nucleotide sequence of the 5' APP promoter. Single-stranded conformational polymorphisms (SSCPs) in the c-FOS gene revealed that c-FOS closely cosegregates with the FAD14 gene but does not show allelic association with FAD. A conservative third-position T-->C mutation was demonstrated in exon 2 (codon 84) of c-FOS, and a C-->G substitution was detected at -209 bp in the 5' promoter of APP. Neither were unique to FAD and are unlikely to be pathogenic or secondary modifiers of the FAD phenotype. We conclude that the c-FOS open reading frame is probably not the site of the FAD14 locus, but we cannot exclude the existence of modifier loci on chromosome 21.

The power of systematic genealogical study in familial Alzheimer disease.

Several kindreds (N, C, To and RB) with familial Alzheimer disease (FAD) from the same small area of Calabria are currently under study. Recently two of us (F.M. and L.F-S.) identified a family in Milan (FJ01) made up of 3 siblings whose parents were of Calabrian origin. Through a subsequent systematic or blanket genealogical study a link has been traced between kindreds To and FJ01. We discuss the relevance of these results to genetic studies.