Biomedical subjects

Gregorio Laino

Publications and source records attributed to Gregorio Laino.

17 recordsLinked to original sources

PubMed2006

An approachable human adult stem cell source for hard-tissue engineering.

Stem cells were obtained from deciduous dental pulp of healthy subjects, aged 6-10 years. This stem cell population was cultured, expanded, and specifically selected, detecting using a FACsorter, c-kit, CD34, and STRO-1 antigen expression. Then, c-kit+/CD34+/STRO-1+ cells were replaced in the culture medium added of 20% FBS, leading to osteoblast differentiation. In fact, these cells, after a week, showed a large positivity for CD44, osteocalcin, and RUNX-2 markers. To achieve an adipocytic differentiation, cells, after sorting, were challenged with dexamethason 10(-8) mM in the same culture medium. To obtain myotube fusion, sorted cells were co-cultured in ATCC medium with mouse myogenic C2C12 cells and, after a week, human stem cell nuclei were found to be able to fuse, forming myotubes. Differentiated osteoblasts, as assessed by a large positivity to several specific antibodies, after 30 days of culture and already in vitro, started to secrete an extracellular mineralized matrix, which, 2 weeks later, built a considerable number of 3D woven bone samples, which showed a strong positivity to alkaline phosphatase (ALP), alizarin red, calcein, other than to specific antibodies. These bone samples, after in vivo transplantation into immunosuppressed rats, were remodeled in a lamellar bone containing entrapped osteocytes. Therefore, this study provides strong evidence that human deciduous dental pulp is an approachable "niche" of stromal stem cells, and that it is an ideal source of osteoblasts, as well as of mineralized tissue, ready for bone regeneration, transplantation, and tissue-based clinical therapies.

Alkaline Phosphatase↗

PubMed2006

Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair.

It is not known whether cells derived from stem cells retain their differentiation and morpho-functional properties after long-term cryopreservation. This information is of importance to evaluate their potential for long-term storage with a view to subsequent use in therapy. Here, we describe the morpho-functional properties of dental pulp stem cells (SBP-DPSCs), and of their differentiated osteoblasts, recovered after long-term cryopreservation. After storage for 2 years, we found that stem cells are still capable of differentiation, and that their differentiated cytotypes proliferate and produce woven bone tissue. In addition, cells still express all their respective surface antigens, confirming cellular integrity. In particular, SBP-DPSCs differentiated into pre-osteoblasts, showing diffuse positivity for ALP, BAP, RUNX-2, and calcein. Recovered osteoblasts expressed bone-specific markers and were easily recognizable ultrastructurally, with no alterations observed at this level. In addition, after in vivo transplantation, woven bone converted into a 3D lamellar bone type. Therefore, dental pulp stem cells and their osteoblast-derived cells can be long-term cryopreserved and may prove to be attractive for clinical applications.

Adult↗

PubMed2006

First International Meeting on "Stem Cell Applications in the Craniofacial Region".

During the 1st International Meeting on "Stem Cell Applications in the Craniofacial Region" promoted in Naples (Italy), invited researchers presented theirwork and the most innovative methods regarding stem cells (SCs) and their application to the craniofacial region of the human body. In addition, some researchers showed their case-reports on craniofacial reconstruction using either osteo-distraction or reconstruction surgical methods. The aim of this biannual meeting is to stimulate discussion, improve knowledge and promote scientific collaboration among basic and clinical scientists in the main topics of SC use in therapy. A summary of this meeting is given.

Craniofacial Dysostosis↗

PubMed2006

In vitro bone production using stem cells derived from human dental pulp.

To harvest bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. The authors describe their preliminary results in this new field with its potential application in craniomaxillofacial surgery. Dental pulp was gently extracted from 34 human permanent teeth (all third molars) of patients 19 to 37 years of age. After they were digested, the cells were selected using a cytometer for c-kit, STRO-1, CD34, CD45, and then for CD44 and RUNX-2. This study, made on a considerable number of cases, provided evidence that dental pulp is extremely rich in stem cells, which were c-kit+/CD34+/STRO-1+/CD45-, capable of differentiation toward several stromal-derived differentiated cells and mainly osteoblasts. These findings, supported by the large number of cases, are of great interest for tissue regeneration, tissue-based clinical therapies, and transplantation.

Adolescent↗

PubMed2005

Genetic effects of anorganic bovine bone (Bio-Oss) on osteoblast-like MG63 cells.

Bio-Oss (Geistlich, Wolhusen, Switzerland) is composed by anorganic bovine bone and is widely used in several bone regeneration procedures in oral surgery. How this biomaterial alters osteoblast gene expression to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identify genes that are differentially regulated in osteoblasts exposed to Bio-Oss. By using DNA microarrays containing 20,000 genes, we identified in osteoblast-like cells line (MG-63) cultured with Bio-Oss several genes which expression was significantly up- and down-regulated. The differentially expressed genes cover a broad range of functional activities: (a) signaling transduction, (b) transcription, (c) cell cycle regulation, (d) vesicular transport, (e) apoptosis, and (f) immunity. These results could explain the reported bioaffinity of Bio-Oss to host animals, its biological affinity to osteogenic cells and its capability to stimulate osteoblastic differentiation. The data reported are, to our knowledge, the first genetic portrait of Bio-Oss effects. They can be relevant to our improved understanding of the molecular mechanism underlying bone regenerative procedures and as a model for comparing other materials with similar clinical effects.

Animals↗

PubMed2005

P-cadherin expression and survival rate in oral squamous cell carcinoma: an immunohistochemical study.

BACKGROUND: P-cadherin (P-cad) is a transmembrane molecule involved in the cell-cell adhesion and similar to E-cadherin (E-cad), but less investigated in oncology, especially in in vivo studies. Aims of the present study were to assess the prevalence of P-cad expression in oral squamous cell carcinoma (OSCC) and to verify whether P-cad can be considered a marker of prognosis in patients with OSCC. METHODS: In a retrospective study, a cohort of 67 OSCC patients was investigated for P-cad expression and its cellular localization by immunohistochemistry; some respective healthy margins of resection were similarly investigated as standard controls. After grouping for P-cad expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G), TNM, Staging, and overall survival rate. Univariate and multivariate analyses were performed. RESULTS: 37 cases (55.2%) of OSCC showed membranous/cytoplasmic positivity for P-cad, whereas 30 (44.8 %) were negative. Although with some differences in membranous vs cytoplasmic localization of P-cad in OSCC with different G, no statistical association was found between P-cad expression and any variables considered at baseline. In terms of prognostic significance, P-cad non expression was found to have an independent association with poorer overall survival rate than P-cad expressing group (P = 0.056); moreover, among P-cad +ve patients the best prognosis was for those OSCC with membranous (P < 0.0001) than those with cytoplasmic P-cad expression. CONCLUSION: On the basis of these results, it is possible to suggest P-cad as an early marker of poor prognosis. The abnormal or lack of P-cad expression could constitute an hallmark of aggressive biological behavior in OSCC.

Adolescent↗

PubMed2005

A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue (LAB).

UNLABELLED: Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. A new c-kit+/CD34+/CD45- cell population of stromal bone producing cells (SBP/DPSCs) was selected, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44+/RUNX-2+), still capable of self-renewing, and then in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue, which is markedly positive for several bone antibodies. This tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone-containing osteocytes. INTRODUCTION: Recently it has been reported that human dental pulp stem cells (DPSCs) are detectable, in humans, only up to the age of 30 years and that they are able to produce in vitro only sporadic calcified nodules and to form, after transplantation in vivo, a mineralized tissue. MATERIALS AND METHODS: Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. Light microscope, histochemistry, immunofluorescence, and RT-PCR analyses were performed to study both stem and differentiating cells. RESULTS AND CONCLUSIONS: A new c-kit+/CD34+/CD45- cell population of stromal bone producing cells (SBP/DPSCs) has been selected by FACSorting, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44+/RUNX-2+), still capable of self-renewing, and in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue. This new-formed tissue is markedly positive for several antibodies for bone, including osteonectin, bone sialoprotein, osteocalcin, fibronectin, collagen III, and bone alkaline phosphatase (BALP). Cells producing LAB can be stored at -80 degrees C for a long period of time and are an extraordinary source of osteoblasts and mineralized fibrous bone tissue. In this study, we also showed that, in aged humans, stem cells can be detected from their pulps. The produced LAB is a fibrous bone tissue resembling the human bone during mineralization, with an external layer formed by osteoblasts markedly positive for osteocalcin. This newly formed tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone containing osteocytes.

Adult↗

PubMed2005

Effect of Vitamin C on pre-osteoblast gene expression.

Ascorbic acid (AA), also known as Vitamin C, is a cofactor required for the function of several hydroxylases. It is not synthesised in humans and has to be provided by diet. Its absence is responsible for scurvy, a condition related to the defective synthesis of collagen by the reduced function of prolylhydroxylase. AA is also a risk factor for periodontal disease. Recently, it has been shown that AA induces embryonic stem cells to differentiate into osteoblasts. The mechanism by which AA sustains pre-osteoblast proliferation and commitment is mediated through the synthesis of collagen type I, interaction with alpha2- and beta1-integrin, activation of the mitogen-activated protein kinase pathway, and phosphorylation of osteoblast-specific transcription factors. However, the multifunctional role of AA is not fully elucidated. MC3T3-E1 mouse calvaria-derived cell line is a well-defined in vitro model of pre-osteoblast differentiation, and AA is essential for the proliferation and differentiation of MC3T3-E1. By using DNA micro-arrays containing 15,000 genes, we identified several genes in MC3T3-E1 cultured with AA for 24h whose expression was significantly up or downregulated. The differentially expressed genes covered a broad range of functional activities: (1) cell growth; (2) metabolism; (3) morphogenesis; (4) cell death; (5) cell communication. The data reported are, to our knowledge, the first genetic portrait of early stage stimulation of pre-osteoblasts by AA, and may be relevant to better understand the molecular mechanism of pre-osteoblast proliferation and commitment. Elucidation of the molecular mechanism has important clinical implications because it may facilitate the correct use of AA to accelerate bone regeneration.

3T3 Cells↗

PubMed2005

Genetic portrait of mild and severe lingual dysplasia.

Squamous cell carcinoma is the most frequent malignant tumor of the oral cavity and often arises from premalignant lesions. Traditional methods used by the pathologist are subjective and lack the sensitivity to predict accurately which precancers may progress with time. Therefore, it is important to search for markers that may identify progression of premalignant lesions. Microarray technology can be use with this aim. Here, we define the genetic expression profile of lingual dysplasia (DS) progression. By using cDNA microarray containing 19.2K clones and a baseline of 11 normal tissues, we compared 5 mild and 4 severe DS. We identified 270 genes differentially expressed in normal tissue vs. mild DS (i.e. 161 up- and 109 down-regulated) and 181 genes differentially expressed in mild vs. severe DS (i.e. 63 up- and 118 down-regulated). The described genes cover a broad range of functional activities: (a) anti-oxidative, (b) DNA-repair, (c) inflammatory response, (d) cell-adhesion/mobility, (e) extracellular matrix depolymerization, and (f) cell-cycle regulation. The data reported better define DS progression and can help in classifying premalignant lesions.

Adult↗

PubMed2005

An in vitro model for dissecting distraction osteogenesis.

Distraction osteogenesis (DO) is a mechanotransduction process capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Several variables are implicated in DO: magnitude of mechanical strain, distraction rate, and type of distracted bone. The combination of these factors acts on different types of cells inducing apoptosis, cell proliferation, and differentiation. The elucidation of the molecular mechanisms has important clinical implications because it may facilitate the use of recombinant proteins or gene therapy to accelerate bone regeneration. Previous reports have analyzed several molecules such as extracellular matrix proteins, cytokines, bone morphogenetic proteins, hormones, and angiogenic factors. Moreover, a single protein can have multifunctional roles. With such a huge number of mechanical, histologic, cellular, and molecular variables, there is the need to have a cell culture model that enables the selection of the effect of a specific strength to a single cell type at different time points and with or without cytokines. The analysis of the genetic profiling of a cell line cultured on an equibiaxial stretch device has such characteristic. Because there is a recruitment and commitment of preosteoblastic cells during bone lengthening and no previous report has focus on them, the authors used a preosteoblast MC3T3-E1 cell line to detect the early molecular effects of distraction on mesenchymal cells. By using DNA microarrays containing 15,000 clones, the authors identified several genes the expression of which was significantly up- or down-regulated. The differentially expressed genes cover a broad range of biological processes: cell growth, metabolism, morphogenesis, cell communication, response to stress, and cell death. The data reported are the first genetic portrait of stretched preosteoblasts. They can be relevant in the better understanding of the molecular mechanism of DO and as a model for comparing the effect of distraction on different cell lines and primary cultures, rate and strength of distraction, and with or without cytokines.

3T3 Cells↗

PubMed2005

Genetic profiling of central giant cell granuloma of the jaws.

Central giant cell granuloma (CGCG) of the jaws is a central osteolytic lesion characterized histologically by multinucleated giant cells in a background of ovoid to spindle-shaped mesenchymal cells. Whether CGCG is a reactive lesion or a truly benign neoplasm remains undetermined, and the mechanism determining the onset of the disease remains unknown. To have more information regarding the genetic events involved in CGCG, the authors decided to perform an expression profile. Samples were derived from two surgically resected CGCG of the mandible. RNA extracted from a pool of three normal bone tissues was used as control. By using DNA microarrays containing 19,200 genes, the authors identified several genes whose expression was significantly up- or down-regulated. The differentially expressed genes cover a broad range of functional activities: cell cycle regulation; signal transduction; and vesicular transport. It was also possible to detect some genes whose function is unknown. The authors believe the data reported to be the first genetic portrait of CGCG of the jaws. Several markers have been identified that can potentially help in identifying some biological behavior (ie, quiescent versus aggressive lesions), as well as genes whose products could be potentially disease-specific targets for therapy. However, the authors think that more cases are needed, especially those comparing quiescent and aggressive lesions, before the exact profile of CGCG is known.

Gene Expression↗

PubMed2005

Alveolar ridge augmentation: a comparative longitudinal study between calvaria and iliac crest bone grafrs.

Insertion of endosseous implants is often difficult because of lack of supporting bone. In the case of severe atrophy of the jaws, a large volume of autogenous bone can be harvested from the iliac crest and calvaria. Both grafts undergo partial resorption with time, but the rate of bone loss has not been fully elucidated. The aim of this study was to evaluate the alveolar bone height gain (ABHG) obtained with iliac crest and calvaria bone grafts. Twenty-five patients had mandibular bone grafts, 32 had maxillary bone grafts, and 11 had both mandibular and maxillary bone grafts. Measures were made on preoperative, postoperative, and follow-up radiographs. A general linear model was used to evaluate the rate of ABHG plotted against months elapsed from the time of the operation to the time of follow-up. General linear model output showed a statistically significant effect for only the type of donor bone graft (P =.004), with a better ABHG for calvaria. The iliac crest bone grafts lost most of the ABHG in the first 6 months, whereas calvaria bone grafts lost ABHG over a greater interval of time. The type of bone graft is the strongest predictor of ABHG, and calvaria bone graft had a higher stability than did iliac bone graft. However, the gap in ABHG between the 2 grafts tended to decrease over time.

Adult↗

PubMed2004

Survivin as prognostic factor in squamous cell carcinoma of the oral cavity.

A series of 78 cases of oral squamous cell carcinoma was analysed by immunohistochemistry for expression of survivin, a recent apoptosis inhibitor. All cases were positive for survivin expression and were divided into two groups using a system of scores. Disease-specific survival curves were calculated according to Kaplan-Meier algorithm, and log rank test was used to compare survival curves. Then, Cox regression analysis was applied to determine the single contribution of covariates on survival rate. So, Cox analysis allowed us to detect the variables most associated to survival. Among the studied variables, such as grade of differentiation, tumor size, stage, recurrence of disease, lymph node presence, only stage and recurrence of disease were predictors of outcome; however, when we analyzed the survival without considering recurrence (that was the stronger predictor of death), a stepwise Cox analysis showed that Survivin, stage and grade of differentiation are significantly associated to survival, with a higher value for Survivin. These data suggest that survivin expression may identify cases of oral squamous cell carcinoma with more aggressive and invasive phenotype and, therefore, could influence the decision for the therapy at the time of diagnosis.

Adolescent↗

PubMed2004

Calcium sulfate: analysis of MG63 osteoblast-like cell response by means of a microarray technology.

Calcium sulfate (CaS) is an highly biocompatible material that has the characteristic of being one of the simplest as well as one of the synthetic bone-like graft with the longest clinical history, spanning more than 100 years. Solidified or crystallized CaS is very osteogenic in vivo. As the surface CaS dissolves in body fluid, the calcium ions form calcium phosphate that reprecipitates on the surface forming an osteoblast "friendly" environment. How this "friendly" environment alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identified genes that are differently regulated in osteoblasts exposed to CaS. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) cultured with CaS (Surgiplaster, Classimplant, Roma, Italy) several genes that expression was significantly upregulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) lysosomal enzymes production, (c) cell cycle regulation, (d) and signaling transduction. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of CaS effects. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Calcium Sulfate↗

PubMed2004

Genetic profiling of granular cell myoblastoma.

Granular cell tumor (GCT), or granular cell myoblastoma, is a relatively uncommon lesion of the soft tissues. It can occur in any organ, and the tongue is more often affected. GCT has unknown etiology, uncertain histogenesis, and a not always benign nature. Benign myoblastomas are the great majority, but rare malignant lesions have been reported. To have more information regarding the genetic events involved in GCT, the authors decided to perform an expression profile. A sample was derived from a surgically resected GCT of the tongue. RNA extracted from normal tongue (mucosa plus muscle) was used as control. By using DNA microarrays containing 19,200 genes, the authors identified several genes for which expression was significantly up- or down-regulated. The differentially expressed genes cover a broad range of functional activities: (1) signal transduction, (2) cell cycle regulation, and (3) cytoskeleton organization. It was also possible to detect some genes whose function is unknown. The data reported are, to the authors' knowledge, the first genetic portrait of GCT. Mutations in some of the described genes are related to neural alterations and mental diseases, and this fact supports the idea of a neural origin of myoblastoma. Several markers have been identified that will help in identifying the biological behavior (when malignant lesions will be described), as well as the gene whose products could be potentially disease-specific targets for therapy.

Cell Cycle Proteins↗

PubMed2004

P-15 cell-binding domain derived from collagen: analysis of MG63 osteoblastic-cell response by means of a microarray technology.

BACKGROUND: P-15 is an analog of the cell-binding domain of collagen. P-15 has been shown to facilitate physiological processes in a way similar to collagen; to serve as an anchorage for cells; and to promote the binding, migration, and differentiation of cells. METHODS: Expression profiling by DNA microarray is a molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cell line (MG-63) cultured with P-15 several genes whose expression was significantly up- or downregulated. RESULTS: The differentially expressed genes cover a broad range of functional activities: 1) signaling transduction, 2) differentiation, 3) apoptosis, and 4) cell-cycle regulation. It was also possible to detect some genes whose function is unknown. CONCLUSIONS: The data reported are, to our knowledge, the first genetic portrait of P-15 effects. They can help us to better understand the molecular mechanism of osteogenesis and can serve as a model for comparing different cell cultures and/or other materials with similar effect.

Apoptosis↗

PubMed2003

Recent developments in orofacial cleft genetics.

Nonsyndromic cleft of the lip and/or palate (CLP or orofacial cleft) derives from an embryopathy with consequent failure of the nasal process and/or palatal shelves fusion. This severe birth defect is one of the most common malformations among live births. Nonsyndromic CLP is composed of two separate entities: cleft lip and palate (CL+/-P) and cleft palate only (CPO). Both have a genetic background, and environmental factors probably disclose these malformations. In CL+/-P, several loci have been identified, and, in one case, a specific gene has also been found. In CPO, one gene has been identified, but many more are probably involved. Because of the complexity of the genetics of nonsyndromic CLP as a result of the difference between CL+/-P and CPO, heterogeneity of each group caused by the number of involved genes, type of inheritance, and interaction with environmental factors, we discuss the more sound results obtained with different approaches: epidemiological studies, animal models, human genetic studies, and in vitro studies.

Animals↗