SEM, TEM, and IHC Analysis of the Sinus Node and Its Implications for the Cardiac Conduction System.

More than 100 years after the discovery of the sinus node (SN) by Keith and Flack, the function and structure of the SN have not been completely established yet. The anatomic architecture of the SN has often been described as devoid of an organized structure; the origin of the sinus impulse is still a matter of debate, and a definite description of the long postulated internodal specialized tract conducting the impulse from the SN to the atrioventricular node (AVN) is still missing. In our previously published study, we proposed a morphologically ordered structure for the SN. As a confirmation of what was presented then, we have added the results of additional observations regarding the structural particularities of the SN. We investigated the morphology of the sinus node in the human hearts of healthy individuals using histochemical, immunohistochemical, optical, and electron microscopy (SEM, TEM). Our results confirmed that the SN presents a previously unseen highly organized architecture.

Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone.

ZrO2 and Al2O3 substrates were successfully coated by a double layer of a silica-based glass named RKKP, using a low-cost firing technique. RKKP is a glass well known for its bioactivity; therefore, a RKKP coating on Al2O3 or ZrO2, allows to combine the excellent mechanical properties of these strong ceramic substrates with its bioactivity. ZrO2 samples were easily coated using a double layer of RKKP by a simple enamelling technique. To accommodate the thermal expansion coefficient mismatch between Al2O3 and RK K P, this substrate was coated using a multilayered composite approach. All of the coatings were characterised from a morphological and compositional point of view, and an extensive biological evaluation was performed using fresh rat osteoblasts. Osteoblast primary cultures were derived from the trabecular bone of femoral condyles harvested from intact (NB) and osteopenic (OB) rats. After characterisation of their phenotype, osteoblasts were seeded on material samples of ZrO2 or Al2O3 coated with RKKP, and cultured for 7 days. Cell proliferation (MTT test) and cell differentiation (alkaline phosphatase activity) were evaluated at the end of the experiment, to assess osteoblast behaviour in the presence of biomaterials and determine if the results were related to the host bone quality. Results of both materials showed a good level of biocompatibility. In particular, MTT significant higher values were detected in NB cultures on ZrO2-RKKP samples; ALP activity significantly increased in NB cultures on Al2O3-RKKP and in OB cultures on both coated samples.

Evaluation of systemic metal diffusion after spinal pedicular fixation with titanium alloy and stainless steel system: a 36-month experimental study in sheep.

It is known that titanium alloys cause more extensive local metallosis due to fretting corrosion than stainless steel implants. The aim of the present study was to investigate possible systemic metal releases (Ti, Al, V, Cr, Ni) in sheep where L4-L5 were implanted with titanium alloy (Ti6Al4V, ASTM F 136) and stainless steel (AISI 316 L). 16 sheep were used: 8 were implanted with Ti6Al4V and 8 with stainless steel. At 6, 12, 24 and 36 months, the following examinations were performed: histology, atomic absorption spectrophotometry (AAS) and scanning electron microscopy (SEM), on liver, lung, kidney, brain, spleen and lumbo-aortic lymph nodes. Hair, urine and arteria blood samples were also analysed by AAS before implantation and at sacrifices. A histologic and ultrastructural study was performed on peri-implant tissues, too. Particular attention was paid to avoid contamination from dissection instruments or use of containers. In basal and in samples at 6 and 12 months, no metals were found in blood, urine, hair or other target tissues of the animals implanted with either Ti6Al4V or stainless steel. Regarding Al, V, Co and Ni, negative results in all tissues and body fluids were obtained also at 24 and 36 months. On the contrary, Ti traces were found in lumbo-aortic lymph nodes and lungs of one sheep only (10 and 30 ng/g, respectively) at 24 months. At 36 months, a systemic diffusion of Ti was observed in all tissues of both sheep instrumented with Ti6Al4V (2-16.5 ng/g), except for body fluids and hair. Metal research in target tissues by light and SEM micro-probe analysis provided negative results. Current data suggest that the amount of Ti found in organs after stable pedicular fixation is extremely low and not biologically available. This observation would lead us to exclude the hypothesis of any toxic reaction and such a release seems to be due to the passive diffusion through lymphatic fluids. Additional studies are needed to confirm if this long-term release of Ti particles might cause tissue damage.

Isolation and characterization of osteoblast cultures from normal and osteopenic sheep for biomaterials evaluation.

Being very useful in the analysis of bone cell differentiation and activity, osteoblast cultures are also used in the in vitro biocompatibility study of new materials. The aim of this work was to evaluate sheep osteoblast cultures derived from normal and ovariectomized animals, and then to assess the in vitro biomaterial behavior on these cultures, taking into account the quality of bone where orthopedic devices are clinically used. For this purpose, we characterized sheep osteoblast cultures, isolated from iliac crest bone of normal (NB osteoblast culture) and osteopenic after ovariectomy (OB osteoblast culture) sheep. Moreover, we studied cell behavior when cultured on different biomaterials (titanium and two biological glasses, RKKP and AP40). Cell characterization at baseline demonstrated that both cultures (NB and OB) showed normal osteoblastic behavior. On the contrary, osteoblasts derived from osteopenic bone and cultivated on AP40 for 6 days revealed a different behavior in terms of both cell morphology and metabolic activity. Statistical analysis (one-way analysis of variance and Scheffé's post hoc multiple-comparison tests) revealed significant differences in Ca level (p<0.0005), MTT test (p<0.0005) and OC production (p<0.05). These in vitro tests demonstrated that sheep osteoblast cultures can be useful when determining biocompatibility and osteointegration of orthopedic materials, and also when evaluating for the presence of osteoporosis.

The ovariectomised sheep as a model for testing biomaterials and prosthetic devices in osteopenic bone: a preliminary study on iliac crest biopsies.

A histomorphometric and ultrastructural evaluation on sheep iliac bone was performed. Six sheep were ovariectomised (OVX Group) and 6 were left intact (Sham-aged, Control Group). An iliac crest biopsy was performed randomly in 6 animals at the beginning of the study, then, in all the animals, after 12 and 24 months. A significant decrease in trabecular bone volume, trabecular thickness (p<0.0005) and cell volume (p<0.005) was observed in OVX animals. A modest decrease in trabecular number and osteoid thickness together with an increase in trabecular separation were observed in OVX animals at 12 and 24 months. The osteoid volume showed a significant difference (p<0.05) between the groups. In OVX animals, at 12 months, Scanning Electron Microscopy revealed an enlargement of the trabecular space and a progressive replacement of bone matrix with adipose tissue. These signs were accentuated at 24 months. In conclusion, OVX sheep showed a loss of trabecular bone starting at 12 months after ovariectomy. The developed osteopenic state may be considered as a useful tool when doing research on biomaterial osteointegration.

Atomic absorption spectrophotometry (AAS) for the evaluation of metallosis in prostheses and artificial organs: a new approach.

To study the presence of metals in body fluids and tissues after implantation of metallic biomaterials and possible related diseases, a new approach in Atomic Absorption Spectrophotometry (AAS) was developed. This technique was compared to three traditional methods: mineralisation with acid digestion (method A) also known as "wet method", dry ashing (with or without oxygen) (method B); classic Kjeldaal (method C). The new approach (method D) modifies the mineralisation phase and the instrument operating instructions. Al, Na, Cr, K, Ni, Co, Ti, Fe, Hg, Pb, V, Sb and Cu levels were tested with the four methods on bone, muscle, cartilage, skin, brain, lymph nodes, blood, urine, and hair. Test results were checked by the addition method. Results demonstrated a significantly higher percentage of Al, Cr, Ni, Ti and Hg recovery with the new approach. The advantages of method D are no residue, no redox reaction, insignificant loss of analytes and enhanced sensitivity (at ppb level vs ppm of the other methods). This approach should be considered especially when testing heavy metals and complex matrices. Its disadvantages are that it is more time consuming and requires the presence of an operator.

Cytologic diagnosis of Acanthamoeba keratitis. Report of a case with correlative study with indirect immunofluorescence and scanning electron microscopy.

We describe a case of Acanthamoeba keratitis in a 20-year-old woman who wore disposable soft contact lenses. The diagnosis was made initially on the basis of a periodic acid-Schiff-stained corneal smear and subsequently confirmed by scanning electron microscopy and immunofluorescence. The patient was successfully treated with a combination of 0.053% polyhexamethylene biguanide and miconazole. Cytologic study and culture of corneal scrapings is relatively painless and inexpensive and may therefore be used for successful diagnosis and follow-up.

A fatal ketamine poisoning.

An unusual case of death by ketamine overdose is reported. The drug's administration was a homicide for homosexual ends. Authors discuss a rapid and effective solid-phase extraction procedure using Bond-Elute* C18 for Ketamine and Nor-Ketamine detection in biological fluids and tissues with a 75% recovery. The drug analysis was carried out by the means of gas-chromatography (GLC) and gas-chromatography/mass spectrometry (GC-MS). The yield of the procedure for Ketamine was: blood 27.4 micrograms/mL; urine 8.51 micrograms/mL; bile 15.2 micrograms/mL; brain 3.24 micrograms/mL; liver 6.6 micrograms/mL; kidney 3.38 micrograms/mL. Nor-Ketamine was detected in all samples, but not quantified.

[Therapy of atopic dermatitis in children. Study on the use of a desensitizing preparation]. / Sulla terapia della dermatite atopica nel bambino. Studio sull'impiego di un preparato iposensibilizzante.

Children with Atopic Dermatitis have been treated with Histaglobin (gamma globulin + histamine) applied by hypodermic injections. The patients (42) were divided into three groups: the first one (ten cases) aged between 9 and 5 years; the second one (eight cases) between 5 and 2 years and the third one (twenty four cases) aged less than two years. The trial has been carried out in two phases: the first one consisted of nine injections with increasing doses ranging from 1/3 to 1 ampoule with 4-6 day intervals; the second one, "the maintenance period", was made by nine injections of 1 ampoule with increasing intervals from two up to four weeks. The following four parameters were checked along all the therapy period: prurigo, erythema, exudation, scratching lesions. All the listed symptoms have been observed to decrease of intensity mainly during the first phase, until the complete disappearance of some of them; the residual symptoms continued to have a further improvement during the second phase; in most case disappeared completely. Absolutely no troubles of any kind have been noticed. Consequently the Authors consider the desensitization therapy by Histaglobin on the whole satisfying.

[Primary enuresis: 144 cases (clinico-statistical observations and therapeutic considerations]. / Enuresi primaria: 144 casi (Osservazioni clinico-statistiche e considerazioni terapeutiche).

Of 144 cases of enuresis we have studied the distribution according with the age in which the subjects have been treated by a doctor for a first consultation, the clinical form (only night, only day, mixed), the sex. We have examined the familiarity, the associated pathology (encopresis, anorexy), the spontaneous evolution and after therapy. Authors have followed a therapeutic scheme that provides for "conditio sine qua non" a practice in the child to reduce, until suspension, the swallowing of liquids during and after the evening meal and in the previous hours, for the whole period of treatment. Cures employed: a preparation with atrapine, ephedrine (Noxenur) and a preparation of dry extract of rear hypophysis (Disipidin), given in series. Another group of subjects has been treated with mipramine. Authors have reached satisfying results by these means, they employ in any case of enuresis on condition to be over 7 years old, because, at this age, there is a more effective and constant collaboration by the subject. Presented the results of EEG and EMG, too.

[The use of scanning electron microscopy (SEM) and microanalysis for the detection of crystalline or amorphous particles in the pulmonary parenchyma]. / L'impiego della microscopia elettronica in scansione (SEM) e della microanalisi per la ricerca di particolati cristallini o amorfi nel parenchima polmonare.

The suitability of scanning electron microscopy with elemental analysis for silica detection in lung is discussed for autopsy specimens from exposed to silica workers. Control specimens are also carried out, with photographic and methodological documentation for main advantage of this technique pointing out.

[Hyperkinetic syndromes. Therapeutic trials]. / Sindromi ipercinetiche. Tentativi terapeutici.

The Authors report their therapeutic experiences with RO 5/4023 begun in 1969, on some hyperkinetic syndromes. The casuistry includes six cases of facial spasm, three cases of Huntington disease, three cases of benign essential tremor, one case of Gilles de La Tourette disease. The favourable results of the drug are estimated in percentage that in somecases has reached 70%.