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Purpose: Human mesenchymal stem cells (hMSCs) are primary cells capable of differentiating to osteocytic lineage when stimulated under appropriate conditions. This study examined changes in hMSC morphology, proliferation, and gene expression after growth on machined or dual acid-etched (AE) titanium surfaces. Materials and Methods: hMSCs, isolated from adult human bone marrow, were cultured on titanium surfaces. The two specimens of titanium surfaces in this study included machined and AE titanium disks. Cell morphology was evaluated by scanning electron microscopy, and cell proliferation and collagen synthesis were estimated by measuring the amount of 3H-thymidine incorporation into DNA and 3H-proline incorporation into collagen fibers. Alkaline phosphatase (ALP) activity was determined by measuring the release of p-nitrophenol from disodium p-nitrophenyl phosphate. Changes in gene expression for bone morphogenetic protein-2 (BMP-2), Runx2 type II, Osterix (Osx), osteopontin, type I collagen, ALP, osteocalcin, and bone sialoprotein were determined by reverse-transcriptase polymerase chain reaction after 22 days of in vitro culture in osteogenic medium. Results: The two substrates had no significant effects on cell adhesion and proliferation. Morphologic characteristics were observed by scanning electron microscopy. hMSCs on the machined surface spread more and were flatter than cells cultured on the AE surface. Osteopontin mRNA expression was similar on all surfaces, and the other mRNA transcripts were increased in hMSC cultured on AE surface. In particular, BMP-2, Runx2, and Osx, three osteogenic factors that induce the progressive differentiation of multipotent mesenchymal cells into osteoblasts, were expressed more on AE titanium than on machined titanium. Collagen and ALP assays confirmed the highest level of mRNA transcripts correlated with increases in these proteins. Conclusion: These results showed that an AE titanium surface stimulated the expression of markers of osteoblastic phenotype more than a machined titanium surface. [ABSTRACT FROM AUTHOR]

PURPOSE: Cell proliferation and extracellular matrix formation are primary events in bone formation. At the dental implant-tissue interface, implant surface roughness modulates osteoblast functions. The aim of the present in vitro study was to investigate the effect of varying surface roughness of titanium implant material on cell proliferation and mRNA expression of specific markers of osteoblast phenotype. MATERIALS AND METHODS: Primary cultures of osteoblasts derived from human mandibular bone were cultured on titanium surfaces. Three titanium surfaces were studied: machined titanium, microsandblasted titanium, and macro-sandblasted titanium (average surface roughnesses of 0.5 and 3 microm, respectively). Cell morphology was estimated by scanning electron microscope analysis and cell proliferation by measuring the amount of 3H-thymidine incorporation into DNA. mRNA expression of osteonectin, osteopontin, bone sialoprotein (BSP), and Runx2, which are markers of osteoblastic phenotype, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Human osteoblasts cultured on machined titanium spread more and were flatter than cells cultured on rough titanium. All blasted surfaces showed significantly higher DNA synthesis than the machined surfaces. Osteonectin mRNA expression was similar on all surfaces. Other mRNA transcripts were increased in osteoblasts cultured on rough titanium surfaces, particularly the macrosandblasted surface. CONCLUSIONS: An average surface roughness of 3 microm (macro-sandblasted titanium) is more suitable than an average surface roughness of 0.5 microm (micro-sandblasted titanium) in favoring osteoblast differentiation in vitro. [ABSTRACT FROM AUTHOR]

A 2-year-old recently spayed female Rottweiler was referred as an emergency with cardiac tamponade and the presence of an anomalous retrograde flow in the pulmonary artery. Echocardiography and angiography demonstrated a left-to-right aortopulmonary fistula. Clinical history and data indicated a possible infectious aetiology. Antibiotics and heart failure medications were administered for 30 days before intervention. Initial attempt at insertion of an Amplatz occluder by means of a percutaneous catheterization technique was tried but a safe release of the device was judged to be not possible due to the angle and the fragile and irregular margins of the window. A decision was made to proceed with a hybrid technique combining thoracotomy and direct pulmonary artery catheterization. This hybrid approach was successful with resolution of congestive heart failure with only residual mild paraprosthetic leakage. [ABSTRACT FROM AUTHOR]

Poly(L-lactide) (PLLA)/single-walled carbon nanotubes (SWNTs) nanocomposite films were produced using the solvent casting method, and morphological, thermal and mechanical properties were investigated. Biocompatibility was evaluated by using human bone cells, performing adhesion and proliferation studies. The role of single-walled nanotube incorporation and functionalization on PLLA bio-polymers was investigated. Pristine (SWNTs) and carboxylated (SWNTs-COOH) carbon nanotubes were considered in order to control the interaction between PLLA and nanotubes. SWNTs and SWNTs-COOH showed a good dispersion in the polymer matrix and improved the PLLA crystallinity. Thermal, morphological and dynamic-mechanical analyses revealed that carboxylic groups on the tube sidewalls increased compatibility between PLLA and nanostructures. Mechanical properties demonstrated an enhancement related to introduction and functionalization of carbon nanotubes. Biological investigations showed osteoblasts cultured on PLLA/SWNTs-COOH nanocomposites has higher cell adhesion and proliferation than osteoblasts cultured on PLLA and PLLA/SWNTs nanocomposites. These studies suggest that combination of biodegradable polymers and SWNTs opens a new perspective in the self-assembly of nanomaterials and nanodevices for biomedical applications with tunable properties. [ABSTRACT FROM AUTHOR]

Background: Barrier membranes are used to prevent down-growth of the oral mucosa along the root surface and to allow alveolar bone regeneration in guided tissue regeneration. Several studies have demonstrated bone regenerates in the presence of bioabsorbable and non-resorbable membranes, but no studies have compared multiple bioabsorbable barriers to one another and to non-resorbable barriers. This study evaluated the in vitro influence of bioabsorbable and non-resorbable membranes on specific parameters of human osteoblast activity.Methods: Human osteoblasts were cultured on bioabsorbable membranes made of collagen, hyaluronic acid, and poly DL-lactide, and the most common non-resorbable membrane which is made of expanded polytetrafluoroethylene (ePTFE). The osteoblasts were cultured in vitro for 24 hours on barrier membranes in the presence of 3H-thymidine and 3H-proline to study cell proliferation and collagen synthesis. Transforming growth factor-beta1 (TGF-beta1) secretion was evaluated in conditioned media using an ELISA kit.Results: The results showed that collagen and poly DL-lactide stimulated DNA synthesis more than ePTFE and hyaluronic acid. All bioabsorbable membranes significantly increased collagen synthesis and alkaline phosphatase activity. Collagen and hyaluronic acid increased secretion of TGF-beta1, a growth factor involved in bone remodeling.Conclusions: These data suggest bioabsorbable membranes, particularly collagen and hyaluronic acid, may promote bone regeneration through their activity on osteoblasts. [ABSTRACT FROM AUTHOR]

Interaction between extracellular matrix (ECM) and cytokines is thought to be crucial for palatal development. The localization of transforming growth factors (TGFα and TGFβ isoforms) in craniofacial tissues suggests that they carry out multiple functions during development. In the present report, we studied TGFα, TGFβ1, and TGFβ3 expressions and their effects on ECM macromolecule production of normal and cleft palatal fibroblasts in vitro, to investigate the mechanisms by which the phenotypic modulation of fibroblasts occurs during the cleft palate process. The results indicated that, while TGFα mRNA was not evidenced in CLP or normal fibroblasts, a reduced TGFβ1 hybridization signal was detected in CLP fibroblasts. In addition, these secreted more active TGFβ3 than TGFβ1, as evaluated in a biological assay. The CLP phenotype, which differed from the normal one because of its higher PG decorin expression and greater production of GAG and collagen, was further modified by the addition of growth factors. In fact, in CLP fibroblasts, TGFα and TGFβ1 down-regulated PG decorin transcript, TGFβ1 increased collagen and GAG in both cellular and extracellular compartments, and TGFβ3 promoted secretory processes of cells. In conclusion, the data represent the first report in a human model in vitro that TGFβ1 and β3 are differently expressed and are correlated to the CLP phenotype. Thus, strength is given to the hypothesis that TGFβ isoforms are the potential inducers of phenotypic expression in palatal fibroblasts during development and that an autocrine growth factor production mechanism may be responsible for the phenotypic modifications. [ABSTRACT FROM AUTHOR]

A 5-month-old female French bulldog was evaluated for the presence of a heart murmur. Through clinical and echocardiographic evaluations, a severe Type A pulmonary stenosis was diagnosed. Angiography during right ventricular catheterization for valvuloplasty revealed drainage from a persistent left cranial vena cava (PLCVC) into the left atrium; this was confirmed later by contrast echocardiography. This report is the first to describe this anatomical variant of a PLCVC in a dog., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of this work was to improve an aqueous two-phase system methodology for fabrication of coherent microcapsules. Simulated microgravity was investigated as tool to improve the cell cluster morphology in order to increase the overall quality of conformal polymer coatings, while the application of two concentric alginate layers and the use of barium instead of calcium as gelling ion was evaluated. Simulated microgravity enabled improvement of neonatal porcine cell cluster sphericity however the freely floating cells, originated during incubation and often found on the capsule surface, raised immunological concerns. Overall, these technical changes translated into improving quality of microcapsules, in terms of either morphologic aspects or the membrane's functional performance. Preparation procedure did not seem to adversely affect viability of the embodied cells. Moreover, the employed alginates high biocompatibility, per se, would promote a good encapsulated cell engraftment. Minimization of last generation microcapsule's size, made of highly purified alginates, represents a further advance on the new horizons of cell therapy for the treatment of a wide variety of chronic disorders, including insulin-dependent diabetes mellitus., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Recombinant human IGF-1 currently represents the only available treatment option for the Laron Syndrome, a rare human disorder caused by defects in the gene encoding growth hormone receptor, resulting in irreversibly retarded growth. Unfortunately, this treatment therapy, poorly impacts longitudinal growth (13% in females and 19% in males), while burdening the patients with severe side effects, including hypoglycemia, in association with the unfair chore of taking multiple daily injections that cause local intense pain. In this study, we have demonstrated that a single intraperitoneal graft of microencapsulated pig Sertoli cells, producing pig insulin-like growth factor-1, successfully promoted significant proportional growth in the Laron mouse, a unique animal model of the human Laron Syndrome. These findings indicate a novel, simply, safe and successful method for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Skin rejection remains a major hurdle in skin reconstructive transplantation surgery. In fact, 85% of the grafted patients experience at least one episode of acute skin rejection in the first year. It has been observed that Sertoli cells (SC), when co-transplanted with allo- or xenogeneic cell/tissues, can induce graft acceptance in the absence of systemic immunosuppression. A method aimed at significantly prolonging skin allografts in rats transplanted with barium alginate-based microencapsulated xenogeneic porcine SC (SC-MCs) is described. Results demonstrated that intraperitoneal (IP) transplantation of SC-MCs with high cellular viability and function can significantly prolong allogeneic skin grafts when compared to transplantation controls receiving only empty alginate capsules (E-MCs). Lymphocytic infiltration at the skin graft site was not observed in 80% of the SC-MCs transplanted rats and these recipient animals showed a significant increased expression of T regulatory (Tregs) cells when compared to E-MCs transplantation controls. The findings of this report further substantiate the positive therapeutic effects of SC on transplantation technology mediated by Sertoli cell-induced alterations of the host's immune system and indicate new perspectives and new strategies for successful skin tissue allografts., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Sertoli cells (SCs) provide an immunoprotective environment to pancreatic islet grafts for treatment of insulin-dependent diabetes. Aim of this work was to verify whether intraperitoneal graft of SCs, enveloped in barium alginate-based microcapsules, would reverse overt spontaneous diabetes in nonobese diabetic (NOD) mice by eliciting generation of newly formed functional islets β-cells., Methods: Microcapsules were prepared, according to our method, by a mono air-jet device system and thereafter examined as far as (a) SC morphology by light microscopy; (b) SC viability by fluorescence microscopy; (c) SC in vitro function; and (d) SC in vivo function, as quoted by diabetes reversal in the NOD mice, were concerned., Results: SCs containing microcapsules exhibited excellent morphology, viability, and function, and when grafted into the NOD's, they induced stable reversion of the disease in 81% of the cases. The treated mice showed dramatic increase in regulatory T lymphocytes (Treg) when compared with control diabetic NOD's treated with empty capsules only. Histologic examination of pancreata retrieved from the SC-transplanted animals showed total disappearance of insulitis, with appearance of new islets, as shown by immunocytochemistry; restored ability of the islets to produce insulin, glucagon, and somatostatin; and finally, increased expression of key transcriptional factors such as neurogenin 3., Conclusions: SCs, enveloped in barium alginate-based microcapsules, showed no long-term loss of their functional and morphological properties in vitro or in vivo. Xenograft of microencapsulated-SC-induced reversal of spontaneous diabetes in the majority of the treated NOD mice, based on SC-related powerful immunomodulatory and pro-β-cell regeneration properties.

When isolated from the iliac crest human mesenchymal stem cells (hMSC) differentiate into osteoblast-like cells with appropriate stimulation in culture. This in vitro study tested the hypothesis that Biostite and hydroxyapatite (HA) affect proliferation and differentiation of hMSC into osteoblastic cells. Cell proliferation was determined by measuring 3H-thymidine incorporation into DNA and typical markers of osteoblastic phenotype were determined by RT-PCR assay. No differences emerged in cell proliferation cultures with Biostite or hydroxyapatite (HA), but gene expression analysis revealed higher expression of collagen,alkaline phosphatase (ALP), osteopontin and bone sialoprotein (BSP) in the presence of Biostite. TGFb2 production, as assessed by an Elisa kit, and Runx2 expression by RT-PCR, were greater in Biostite cultures, suggesting Biostite provides a better environment for hMSC differentiation into osteoblasts and is, potentially, a more promising bone-filling material than HA.

Purpose: To assess whether prevention of unexpected in vivo adverse inflammatory and immune responses to biohybrid organ grafts for the treatment of Type I Diabetes Mellitus (T1DM) is possible by superoxide dismutase and ketoprofen controlled release., Methods: Superoxide dismutase and ketoprofen-loaded polyester microspheres were prepared by W/O/W and O/W methods, embodied into purified alginate-poly-L-ornithine-alginate microcapsules and intraperitoneally implanted into CD1 mice. The microspheres were characterized for morphology, size, encapsulation efficiency, enzyme activity and in vitro release. Purified alginate contaminants were assayed, and the obtained microcapsules were investigated for size and morphology before and after implantation over 30 days. Cell pericapsular overgrowth and expression were evaluated by optical microscopy and flow cytometry., Results: Superoxide dismutase and ketoprofen sustained release reduced cell pericapsular overgrowth in comparison to the control. Superoxide dismutase release allowed preserving the microcapsules over 30 days. Ketoprofen-loaded microspheres showed some effect in the immediate post-grafting period. A higher macrophage and T-cell expression was observed for the control group., Conclusions: Microspheres containing superoxide dismutase and ketoprofen may represent novel tools to limit or prevent unpredictable adverse in vivo response to alginate, thus contributing to improve cell transplantation success rates in T1DM treatment.

The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM). Starting from isolated neonatal porcine pancreatic islets (NPIs), we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs) for different time periods (7, 14, 21 days). To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation of β-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs β-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature β-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM.

Type I diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells, and effective treatment of the disease might require rescuing beta cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional beta cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-beta-dependent emergence of autoantigen-specific regulatory T cells and recovery of beta cell function in the diabetic recipients.

Tissue infiltration is different in desmoid and fibroma tumours. Both produce high levels of transforming growth factor beta1 (TGFbeta1), which is related to extracellular matrix (ECM) accumulation which in turn regulates cell function and cell migration. Interactions between collagen, proteoglycans and cell surface fibronectin are involved in the assembly and functions of the ECM. As toremifene inhibits collagen and TGFbeta1 synthesis, we tested it in normal, desmoid and fibroma fibroblasts. We will report the changes in glycosaminoglycan (GAG) and collagen synthesis, TGFbeta1 activity, fibronectin mRNA expression and TGFbeta1 receptors after toremifene treatment in normal, fibroma and desmoid fibroblasts. We evaluated GAG and collagen synthesis with 3H-glucosamine and 3H-proline incorporation, TGFbeta1 activity with the ELISA method, TGFbeta1 receptor affinity with 125I-TGFbeta1 binding and total RNA with Northern blot analysis. GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels were higher in fibroma and desmoid than normal fibroblasts. The increase was greater in desmoid than fibroma tumour cells. Toremifene treatment reduced GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels in all cell cultures. The percentage reduction in GAG was similar in all cultures; the reduction in collagen synthesis and TGFbeta1 activity was the highest in desmoid fibroblasts. TGFbeta1 receptors were higher in fibroma and desmoid cells than controls. Toremifene reduced TGFbeta1 receptors only in desmoid fibroblasts, with no effect on the changes in type I, II, and III receptors. Our data show that toremifene modifies the ECM components that regulate cytokine activity and cell migration. The reduction in receptor number only in desmoid cells suggests that toremifene may reduce TGFbeta1's affinity for its receptors. Synthesis of a substance regulating protein kinase activity, which is directly involved in the link between TGFbeta1 and its receptors, cannot be excluded.

A method for microencapsulation of isolated neonatal porcine Sertoli cells is described. Using a conventional alginate-poli-L-ornithine encapsulation procedure, which has been used in our laboratory for almost two decades to envelop pancreatic islets, we observed significant loss of Sertoli cell viability, possibly due to excessive Ca(2+) ion exposure. Replacing calcium with barium, or shortening the incubation period in the presence of Ca ions, we obtained barium or calcium alginate gel microbeads that did not alter morphology and viability of the encapsulated Sertoli cells. The procedure might permit access to a novel approach to immunologically alter cell graft acceptance.

Desmoid and fibroma tumours are characterized by cell proliferation, glycosaminoglycan and collagen fibre accumulation, high levels of transforming growth factor beta(1) (TGFbeta(1)) and different patterns of tissue infiltration. TGFbeta(1) is related to extracellular matrix (ECM) composition which, in turn, regulates cell functions and cell migration. In this study we report changes in cell proliferation, glycosaminoglycan (GAG) and collagen synthesis, TGFbeta(1) mRNA expression and fibronectin levels in normal, desmoid and fibroma fibroblast cultures before and after TGFbeta(1) stimulation. Our data showed cell proliferation, GAG and collagen synthesis, transforming growth factor beta(1) mRNA expression and fibronectin levels were significantly higher in desmoid than in fibroma cultures. TGFbeta(1) treatment had no effect on cell proliferation, but increased TGFbeta(1) mRNA expression, GAG, fibronectin and collagen synthesis in desmoid and fibroma fibroblasts. Its effects were more marked in desmoid cells. Fibronectin favours cell migration, while changes in GAG composition alter cell behaviour and ECM organization. In conclusion our data suggest that the different patterns of infiltration in desmoid and fibroma tumours are due to changes in ECM components and cell-ECM interactions which can be ascribed to altered TGFbeta(1) mRNA expression and TGFbeta(1) activity.

Normal branching development is dependent on the correlation between cells and extracellular matrix. In this interaction glycosaminoglycans, cytokines and growth factors play a fundamental role. In order to verify the distribution and influence of extracellular matrix and related enzymes on chick embryo lung development, 6 day-old whole lungs were maintained in vitro with testicular hyaluronidase, beta-N-acetyl-D-glucosaminidase and chondrotinase ABC or in linkage with apical, medial and caudal lung regions of 6-day development before and after enzyme treatment. In a separate lung region beta-N-acetyl-D-glucosaminidase and hyaluronidase were determined. Our data show that the whole lung cultures increase bronchial branching development when the medial region is admixed separately, while the separate apical or caudal regions or apical combined with caudal region do not affect bronchial branching development. The enzyme treatment of medial region prevents the branching development in associated whole lung. The bronchial branching development of whole lung cultured in medium containing the enzymes related to glycosaminoglycans turnover is significantly altered. In conclusion, these data show that the different influence of separate apical, medial, caudal lung regions on bronchial branching development is related to the extracellular matrix composition.

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.

During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-beta binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA--which, at pharmacologic doses, induces cleft palate in newborns of many species--were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-beta signaling systems could be involved in human cleft palate fibroblast phenotype.

Background: The restricted availability of cadaveric human donor pancreases mandates validation of possibly inexhaustible, alternative sources of insulin secretory cells in order to expand islet transplant for the therapy of insulin dependent diabetes mellitus (T1DM)., Methods: Neonatal pig pancreatic islets (NPI), isolated and purified by our method, were specially cultured until confluent cell monolayers were obtained. Expression of several beta-cell phenotype transcriptional factors, under glucose and other stimuli, were examined throughout 90 days of culture., Results: High glucose concentration and glucagon-like peptide 1 (GLP-1) were associated with maintenance either of insulin secretory patterns from the incubated cell monolayers, or expression of transcriptional markers associated with beta-cell like phenotypes., Conclusion: Morphological and molecular expression of beta-cell markers and products from NPI cell monolayers seem to identify a novel and potentially powerful source of insulin producing cells that might fulfill transplant needs for insulin substitution therapy.

Immunological features of GM-490 cells, a new blood cell line from a patient with acute lymphoblastic leukemia, included lack of CD34, CD38, CD45, CD14, HLA-DR, and lymphoid and myeloid markers and expression of CD29, CD36, CD44, CD54, CD71, CD105, and CD133. Molecular analysis indicated CD45 gene expression was absent but CD34 mRNA was present. GM-490 cells constitutively produced fibronectin (FN), type III and traces of type I collagen, collagenases, glycosaminoglycans (GAG) and biglycan and betaglycan proteoglycans (PG) as well as FGF2 and TGFbeta1. When FGF2 and/or TGFbeta1 were added to cells in vitro, they stimulated cell proliferation and differently modulated matrix production and growth factor receptor expression. Reverse transcription-polymerase chain reaction (RT-PCR) detection of transcripts encoding for osteocalcin and RUNX2 suggests GM-490 cells differentiate towards the osteoblast pathway. GM-490 cells expressed the low affinity nerve growth factor receptor (p75LNGFR), a somatic stem cell marker that is not detected in hematopoietic cells, leading to the hypothesis that GM-490 has mesenchymal stem cell properties. The reciprocal modulating effects of FGF2 and TGFbeta1 on each other's receptors make the GM-490 cell line a new model for investigating the relationship between these growth factors and their receptors in autocrine loops which are believed to sustain the malignant clone in hematological diseases., (Copyright 2005 Wiley-Liss, Inc.)

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of beta-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation beta-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

Background: Bone marrow mesenchymal stem cells (MSCs) are currently being investigated in preclinical and clinical settings because of their multipotent differentiative capacity or, alternatively, their immunosuppressive function. The aim of this study was to evaluate dental pulp (DP) as a potential source of MSCs instead of bone marrow (BM)., Methods: Flow cytometric analysis showed that DP-MSCs and BM-MSCs were equally SH2, SH3, SH4, CD29 and CD 166 positive. The in vitro proliferative kinetics of MSCs were measured by 3H-thymidine incorporation uptake. The immunosuppressive function of MSCs was then tested by coculturing PHA-stimulated allogeneic T cells with or without MSCs for 3 days., Results: BM-MSCs could be differentiated in vitro into osteogenic, chondrogenic and adipogenic lineages. DP-MSCs showed osteogenic and adipocytic differentiation, but did not differentiate into chondrocytes. Although DP-MSCs grow rapidly in vitro between day 3 and day 8 of culture and then decrease their proliferation by day 15, BM-MSCs have a stable and continuous proliferation over the same period of time. The addition of DP-MSCs or BM-MSCs resulted in 91 +/- 4% and 75 +/- 3% inhibition of T cell response, respectively, assessed by a 3H-thymidine assay., Conclusions: Dental pulp is an easily accessible and efficient source of MSCs, with different kinetics and differentiation potentialities from MSCs as isolated from the bone marrow. The rapid proliferative capacity together with the immunoregulatory characteristics of DP-MSCs may prompt future studies aimed at using these cells in the treatment or prevention of T-cell alloreactivity in hematopoietic or solid organ allogeneic transplantation.

Apert and Crouzon syndromes are well known craniostenosis. In the last 10 years several studies were performed to provide a better understanding of the etiology and pathogenesis of these diseases. Both have an autosomal dominant mode of transmission, and a mutation in the gene encoding for the fibroblast growth factor receptor 2 (FGFR2) is the cause in most patients. However, the fact that the same mutation can produce a wide range of phenotypic expression makes the mechanism of anomalous development more complex. The extracellular matrix (ECM) is composed of proteins, glycosaminoglycans, and cytokines that are secreted in an autocrine and paracrine manner and are able to modify the ECM. Fibroblast growth factors are complexed with heparan sulfate, a component of the ECM, before binding the FGFR2. Data exist about different expressions of cytokines and ECM macromolecule in craniostenosis-derived fibroblasts and osteoblasts. Changes in ECM composition could explain the altered osteogenic process and account for pathologic variations in cranial development in addition to the FGFR2 mutations.

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/ differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the beta-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from < 3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in beta-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional beta-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.

Crosslinking of collagen biomaterials increases their resistance to degradation in vivo. Glutaraldehyde (GA) is normally used to crosslink collagen biomaterial, but is often cytotoxic. Diphenylphosphoryl azide (DPPA) has recently been proposed as reagent, but little is known about its effects on cell behavior. In this study, we determined which collagen membrane was the most biocompatible: Paroguide which is crosslinked with DPPA and contains chondroitin sulfate; Opocrin which is crosslinked with DPPA; Biomed Extend which is crosslinked with GA; and Bio-Gide which is left untreated. Cell proliferation and extracellular matrix macromolecule deposition were evaluated in human fibroblasts cultured on the membranes. The GA-crosslinked Biomed Extend membrane and the not-crosslinked Bio-Gide membrane reduced cell growth and collagen secretion compared with DPPA-crosslinked biomembranes. When Paroguide and Opocrin were compared, better results were obtained with Paroguide. The greatest amount of transforming growth factor beta1, a growth factor involved in extracellular matrix macromolecule accumulation and in tissue regeneration, was produced by cells cultured on Paroguide, with Opocrin second. Our data suggest that the DPPA method is more biocompatible than the GA for crosslinking collagen biomaterials and that membranes made of collagen plus chondroitin sulfate are better than membranes made of pure collagen., (Copyright 2003 Wiley Periodicals, Inc.)