Human CD34+ stem cells produce bone nodules in vivo.

OBJECTIVES: The aim of this study was to select and provide enough stem cells for quick transplantation in bone engineering procedures, avoiding any in vitro expansion step. MATERIALS AND METHODS: Dental germ pulp, collected from 25 healthy subjects aged 13-20 years, were subjected to magnetic-activated cell sorting to select a CD34(+) stem cell population capable of differentiating into pre-osteoblasts. These cells were allowed to adhere to an absorbable polylactic-coglycolic acid scaffold for 30 min, without any prior expansion, and the CD34(+) cell-colonized scaffolds were then transplanted into immunocompromised rats, subcutaneously. RESULTS: After 60 days, analysis of recovered transplants revealed that they were formed of nodules of bone, of the same dimensions as the original scaffold. Bone-specific proteins were detected by immunofluorescence, within the nodules, and X-ray diffraction patterns revealed characteristic features of bone. In addition, presence of platelet endothelial cell adhesion molecule and von Willebrand factor immunoreactivity were suggestive of neo-angiogenesis and neovasculogenesis taking place within nodules. Importantly, these vessels were HLA-1(+) and, thus, clearly human in origin. CONCLUSIONS: This study indicates that CD34(+) cells obtained from dental pulp can be used for engineering bone, without the need for prior culture expanding procedures. Using autologous stem cells, this schedule could be used to provide the basis for bone regenerative surgery, with limited sacrifice of tissue, low morbidity at the collection site, and significant reduction in time needed for clinical recovery.

Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.

Stromal stem cells from human dental pulp (SBP-DPSCs) were used to study osteogenic differentiation in vitro and in vivo. We previously reported that SBP-DPSCs are multipotent stem cells able to differentiate into osteoblasts, which synthesize three-dimensional woven bone tissue chips in vitro. In this study, we followed the temporal expression pattern of specific markers in SBP-DPSCs and found that, when differentiating into osteoblasts, they express, besides osteocalcin, also flk-1 (VEGF-R2). In addition, 30% of them expressed specific antigens for endothelial cells, including CD54, von-Willebrand (domain 1 and 2), CD31 (PECAM-1) and angiotensin-converting enzyme. Interestingly, we found endotheliocytes forming vessel walls, observing that stem cells synergically differentiate into osteoblasts and endotheliocytes, and that flk-1 exerts a pivotal role in coupling osteoblast and endotheliocyte differentiation. When either SBP-DPSCs or bone chips obtained in vitro were transplanted into immunocompromised rats, they generated a tissue structure with an integral blood supply similar to that of human adult bone; in fact, a large number of HLA-1+ vessels were observed either within the bone or surrounding it in a periosteal layer. This study provides direct evidence to suggest that osteogenesis and angiogenesis mediated by human SBP-DPSCs may be regulated by distinct mechanisms, leading to the organization of adult bone tissue after stem cell transplantation.

Bone grafting with platelet-rich plasma in alveolar cleft. Case report.

Bone grafting of the alveolus has become an essential part of the contemporary surgical management of oral clefts. The benefits of this procedure are the stabilization of the maxillary arch, elimination of oronasal fistulae, the reconstruction of the soft tissue nasal base support, creation of bony support for subsequent tooth eruption or, when they are not present or not preserved, for implants application. The authors show a case of bone grafting with the aid of platelet-rich plasma (PRP). Because of the difficulties due to the oral cleft and to its surgical reparation (big size of bone defect, hard scars and sclerotic soft tissue) the authors decided to add PRP to a bone graft taken from the chin. PRP contains a high concentration of growth factors and is able to stimulate both wound and bone regeneration. Infact, the authors have observed very good results both in bone integration and in soft tissue reparation.

Effects of Emdogain on osteoblast gene expression.

OBJECTIVE: Emdogain (EMD) is a protein extract purified from porcine enamel and has been introduced in clinical practice to obtain periodontal regeneration. EMD is composed mainly of amelogenins (90%), while the remaining 10% is composed of non-amelogenin enamel matrix proteins such as enamelins, tuftelin, amelin and ameloblastin. Enamel matrix proteins seem to be involved in root formation. EMD has been reported to promote proliferation, migration, adhesion and differentiation of cells associated with healing periodontal tissues in vivo. DESIGN: How this protein acts on osteoblasts is poorly understood. We therefore attempted to address this question by using a microarray technique to identify genes that are differently regulated in osteoblasts exposed to enamel matrix proteins. RESULTS: By using DNA microarrays containing 20,000 genes, we identified several upregulated and downregulated genes in the osteoblast-like cell line (MG-63) cultured with enamel matrix proteins (Emd). The differentially expressed genes cover a broad range of functional activities: (i) signaling transduction, (ii) transcription, (iii) translation, (iv) cell cycle regulation, proliferation and apoptosis, (v) immune system, (vi) vesicular transport and lysosome activity, and (vii) cytoskeleton, cell adhesion and extracellular matrix production. CONCLUSIONS: The data reported are the first genome-wide scan of the effect of enamel matrix proteins on osteoblast-like cells. These results can contribute to our understanding of the molecular mechanisms of bone regeneration and as a model for comparing other materials with similar clinical effects.

Potential markers of tongue tumor progression selected by cDNA microarray.

Squamous cell carcinoma (SCC), the most frequent malignant tumor of the oral cavity, generally exhibits a poor prognosis and metastases are the main cause of death. This tumor often arises from pre-malignant lesions. To date, it is difficult to predict if and which pre-malignant lesions may progress into oral SCC using traditional methods. For these reasons, several studies are trying to identify markers useful in the progression of pre-malignant lesions and tumors. To define the genetic expression profile of tongue tumor progression we compared 9 dysplasias (DS), 8 tumors without metastasis (TWM), 11 metastasizing SCCs (MT) of the tongue, and a baseline of 11 normal tissues by using cDNA microarray containing 19.2 K clones. We initially applied hierarchical agglomerative clustering based on information from all 6026 clones. Results were obtained by performing a two steps analysis: a Significance Analysis of Microarray (SAM) and a Gene Ontology search. One hundred and five clones have statistically significant different expression levels (FDR < 0.01) between DS and TWM, whereas 570 genes have statistically significant difference expression levels between TWM and MT (FDR < 0.01) as detected by SAM. By filtering with FatiGo only 33 genes were differentially expressed in TWN, respect to DS, whereas 155 genes were differentially expressed in MT respect to TWM. We detected some genes which encode for oncogenes, transcription factors and cell cycle regulators as potential markers of DS progression. Examples are BAG4, PAX3 and CCNI, respectively. Among potential markers of metastases are some genes related to cell mobility (TSPAN-2 and SNTA1), intercellular adhesion (integrin alpha 7) or extracellular matrix components (ADAMTS2 and cathepsin O). Additionally, under-expressed genes encoded apoptosis-related proteins (PDCD4 and CASP4). In conclusion, we identified several genes differentially expressed in tumor progression which can potentially help in better classifying pre-malignant lesions and tongue SCCs.

Orofacial cleft in Southern Italy.

AIM: Cleft lip and palate or orofacial cleft (OFC) is one of the most common congenital malformations. The average incidence is around 1 every 1 000 live births. Different types of cleft lip and palate exist: cleft lip (CL), cleft lip and alveolus (CLA), cleft lip, alveolus and palate (CLP), and cleft palate only (CPO). Genetic studies on human samples have demonstrated that OFC has a heterogeneous genetic background and environmental factors also contribute to disclose this malformation. Because of the complex aetiology of OFC, studies on different and homogeneous populations can be useful in detecting environmental and genetic factors involved in the onset of this disease. The aim of the present study is to evaluate the relation between gender, type of cleft and affected side in a group of patients in Southern Italy. METHODS: Six hundred and fifty patients were enrolled in this retrospective study. They were operated at the Dental Clinic of the Second University of Naples in the period 1980-2002. Gender, type of cleft and affected side were analysed by means of the "Test for comparing two proportions". RESULTS: Among the analysed variables it was statistically demonstrated that overall CLP is more frequent in males as well as bilateral CLP whereas overall CPO is more frequent in females as well as right microform of CL. CONCLUSION: The identification of gender related subtypes of cleft is in accordance with data reported in similar studies on different populations and confirms that OFC is an heterogeneous disease even in a homogeneous ethnic group.

Benign fibrous histiocytoma (BHF) of the cheek: CD 68-KP1 positivity.

We present a case of fibrous histiocytoma of the cheek in a 32-year-old male with no evidence of any regional invasion or distant metastasis. Pathologic analysis and diagnosis of these lesions is often challenging, and usually based on a combination of light microscopy and immunohistochemistry. In this study the diagnosis was confirmed using immunostaining with the antibody CD 68-KP1 that is positive in any lesion containing lysosomal granules or phagolysosomes.

[Experimental liver transplantation in pigs. Surgical technique and complications]. / Trapianto di fegato sperimentale nel maiale. Tecnica chirurgica e complicanze.

Only recently, in our laboratory of experimental surgery, we started with a protocol for orthotopic liver transplantation (OLT) in a pig model. This was felt as mandatory for experimental purposes as well as for future clinical applications at our center. We report herein our own experience with 41 OLTx. Intraoperative "lethal" complications occurred in up to 32% (14/41) whereas postoperative complications occurred in the remainders at different intervals of time with a maximum survival of 30 days. No attention was paid to prevent rejection-infection episodes. The main cause of death was the primary non-function (PNF) or dis-function (PDF) manifested either intra or postoperatively in 16 out the 41 OLTx (39%). Intraoperative technical errors accounted for up to 9% (4/41 OLTx). Acute hemorrhage gastritis and gastric perforations occurred postoperatively in 6 animals (14%) and represent one of the peculiar aspects of OLT in pig model.

Protection of rat heart from ischaemia-reperfusion injury by the 21-aminosteroid U-74389G.

In ischaemia-reperfusion syndromes lipid peroxidation appears an important factor contributing to tissue damage. The 21-aminosteroids (lazaroids) exhibit beneficial effects in various pathological conditions, especially in post-traumatic lesions of the central nervous system, where a peroxidative injury seems to be involved. The aim of our study was to ascertain if one of these compounds, U-74389G, plays a significant role in protecting heart muscle from ischaemia-reperfusion damage. Rat hearts used for heterotopic transplantation represented the experimental model in this investigation. Animals (Wistar rats weighing 200-250 g) were divided into five groups: controls, untreated and treated donors, untreated and treated recipients. Donors were anaesthetized and heparinized, and the heart was excised through a bilateral thoracotomy, arrested with St Thomas solution and stored in cold saline for 2 hours. For the recipient preparation, a modified Ono's technique was used, and heart reimplantation was performed with a termino-lateral aorto-aortic anasthomosis and a termino-lateral pulmonary-cava anasthomosis. After the anasthomoses were completed hearts were reperfused for 30 min; then hearts were excised and specimens were taken for biochemical and morphological studies. These were conducted on three groups of hearts: (A) hearts reimplanted and reperfused without treatment of the donor or of the recipient animal; (B) hearts subjected to the same procedure but in the presence of U-74389G treatment of donors and recipient rats; (C) control hearts rapidly excised from normal, non-operated animals. Electron microscopy studies showed, in hearts transplanted without treatment, the typical morphological aspects of lipoperoxidative injury: swollen mitochondria with disrupted cristae, damaged endothelial cells with the nucleous bulging into the lumen and a discontinued endothelial lining with diffuse oedema among the fibers. Lazaroid treatment attenuated most of these damages in hearts of group B. As for the biochemical findings, the hearts transplanted in the presence of U-74389G treatment had significantly higher ATP and creatine phosphate levels (P < 0.01) and lower malondialdehyde concentrations (P < 0.05) with respect to the hearts transplanted without treatment. Furthermore, serum creatine kinase activity was lower in treated than in untreated recipient animals (P < 0.05). Taken together, all these results indicate that U-74389G treatment is effective in protecting cardiac muscle from structural and functional ischaemia-reperfusion injuries, at least from those arising during a heart transplantation procedure.

[Surgical staging of pulmonary carcinoma. Probability of error]. / Stadiazione chirurgica del carcinoma polmonare. Probabilità di errore.

234 patients with lung cancer and operated in Thoracic and Cardiovascular Surgery Department of Careggi Hospital in Florence have been evaluated in order to examine surgical staging accuracy in comparison with pathological staging. There is a statistically significative difference between surgical and pathological staging as a datum point. Surgeon is inclined to over-estimate the lymph-nodes involvement and the primitive tumor extension. It is important to bear in mind this bent whenever decisions of surgical strategy have to be taken.

["N2" carcinoma of the lung. Therapeutic considerations]. / Carcinoma del polmone "N2". Considerazioni terapeutiche.

The follow-up of 240 N2 lung cancer cases operated in Thoracic and Cardiovascular Surgery Department of Florence is examined. The analysis is performed in compliance with global survival, "T", histology, therapeutic choices. Global survival is 81% after 6 months, 60% after 1 year, 37% after 2 years, 26% after 3 years, 23% after 4 years, 23% after 5 years. Significative difference on survival does not exist between principal histologic types (squamous, adenocarcinoma, adenosquamous). Raising the "T" survival decreases, but only for adenocarcinoma. Different therapeutic options (only surgery, surgery+radiotherapy, surgery+chemotherapy, surgery+radio and chemotherapy) do not influence the survival in a way statistically significative. From the literature, any certainty about radiotherapy and chemotherapy associated to surgery for N2 lung cancer treatment does not exist at the moment. Thus radical surgery is essential.

[Immigration areas and urban examples: the case of Naples]. / Espaces d'immigration et formes urbaines: considerations sur le cas de Naples.

PIP: This is a case study on the problems faced by third world immigrants in Europe, using the example of housing problems experienced by immigrants from north Africa to Naples, Italy.^ieng

Gastric bicarbonate secretion in high-relapsing, smoking duodenal ulcer patients.

Gastric bicarbonate secretion has been evaluated by Feldman's method in 48 duodenal-ulcer patients. The relationship between smoking, clinical ulcer outcome (healing and recurrence) and bicarbonate secretion has been analysed. Heavy smokers secreted higher bicarbonate ions than did non-smokers. High-relapsing patients produced lower bicarbonate output. These preliminary data suggest that an impaired gastric bicarbonate secretion is associated with smoking, a well-known ulcer-associated factor; further-more, it may single out high-relapsing duodenal ulcer patients.

[Alcohol-dependent mast cell activation in ulcer]. / Attivazione delle mastcellule alcol-dipendente nella malattia ulcerosa.

Numerous studies have shown that alcohol causes both acute and chronic damage to gastroduodenal mucosa. The methods of damage differ however, and experimental studies in animals have shown that the degranulation of mast cells in gastric mucosa causes acute hemorrhagic lesions after the consumption of alcohol. It is not known whether this mechanism also operates in man. The aim of the present study was therefore to evaluate whether there is a correlation between mast-cell activation, determined by assaying tryptase levels in gastric mucosa, and the consumption of alcohol in patients with ulcerative diseases. Thirty-one patients with cicatrized ulcerative lesions (13 gastric ulcers, 18 duodenal ulcers) were included in the study. Biopsies at the level of the gastric fundus and antrum and the duodenal bulb were performed in all patients to determine tryptase levels. Biopsy material was frozen and subsequently homogenized; the enzyme was assayed in the supernatant using a radioimmunometric method. The mean daily alcohol consumption was calculated in clinical terms for each patient over the past 5 years and patients were subdivided into non-drinkers and moderate (< 60 g alcohol/day) and excessive (> 60 g alcohol/day) drinkers. It was found that tryptase concentrations were higher in the fundus compared to the gastric antrum and duodenal bulb, irrespective of alcohol consumption both in patients with gastric ulcer and duodenal ulcer. The importance of mast cells in provoking alcohol-dependent damage was studied at a gastric level. Alcohol leads to their degranulation and therefore contributes to the formation of gastric lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Esaprazole effect on acid, peptic and alkaline secretion in duodenal ulcer patients.

To study the effect of Esaprazole, a new antiulcer drug, on acid, peptic and alkaline secretion a modified gastric acid test was performed in 18 duodenal ulcer patients. Pentagastrin was administered as bolus 30' and 75' after the beginning of the test, followed by Esaprazole 300 mg i.v. at 90'. Gastric juice was collected every 15' for determination of: total volume, volume of non parietal secretion, acid, bicarbonate and pepsin output. Serum pepsinogen group I was determined by radioimmunoassay. Esaprazole had a significant inhibitory effect on the total volume of gastric secretion and on volume of non parietal secretion. Pepsin output and serum pepsinogen group I were not affected by Esaprazole, while bicarbonate secretion was reduced. Antiulcer activity of Esaprazole seems to be due to the reduction of total volume of gastric secretion.