Tissue reaction to sealing materials: different view at biocompatibility

The biodegradability of root canal sealers in areas other than the root canal system is crucial to the overall success rate of endodontic treatment. The aim of the present study was to investigate, the cell and tissue reaction to GuttaFlow and AHPlus, both in vitro and in vivo. For the in vitro experiments the materials were incubated with Human Periodontal Ligament Fibroblasts and cell proliferation and cytotoxicity analyses were performed. Additional fluorescence-microscope stainings were carried out in order to visualize cell growth and morphology. For assessment of the tissue reaction to the materials a subcutaneous implantation model in Wistar rats was employed and the inflammatory response to the materials was visualized by means of general and specific histology after 6 weeks. Human gingival fibroblasts proliferation seemed to be dependent upon dental material and cultivation time. After an incubation period of 96 hrs AHPlus proved to be significantly (p < 0.002) more cytotoxic than GuttaFlow, as only a small number of fibroblasts survived on AHPlus. In vivo, GuttaFlow was surrounded by a fibrous capsule and no degradation took place, while AHPlus induced a well-vascularized granulation tissue in which the material was phagocyted by macrophages. The results of this study demonstrate that a potential cytotoxic effect of a sealing material may beneficial in order to have antibacterial properties and induce self degradation when accidentally extruded over the apical foramen

PTCH-1 and MDM2 expression in ameloblastoma from a West African sub-population: Implication for chemotherapeutics

Introduction: Ameloblastoma is a slow growing, painless odontogenic swelling which can attain sizes that result in severe deformities of thecraniofacial complex. It is the most commonly encountered odontogenic tumor in Nigeria. Surgical intervention is currently the method of treatment; however identification of altered molecular pathways may inform  chemotherapeutic potential. The Protein Patched homolog 1 (PTCH-1)is overexpressed in ameloblastoma. Also, mutation in the MDM2 gene can reduce the tumor suppressor function of p53 and promote ameloblastoma growth. No study however has characterized the molecular profile of African cases of ameloblastoma with a view to developing chemotherapeutic alternatives. The objective was to characterize the PTCH-1 genetic profile of Ameloblastoma in Nigerian patients as a first step in investigating its potential for chemotherapeutic intervention.Methods: Twenty-eight FFPE blocks of ameloblastoma cases from Nigerian patients were prepared for antibody processing to PTCH-1 (Polyclonal  Anti-PTCH antibody ab39266) and MDM2 (Monoclonal Anti-MDM2 antibody (2A10) ab16895). Cytoplasmic brown staining was considered as positive for PTCH while nuclear staining was positive for MDM2.Results: Moderate and strong expressions for PTCH in ameloblast and stellate reticulum were 78.6% and 60.7% respectively. Only 3 (10.7%) cases expressed MDM2.Conclusion: The importance of our study is that it supports, in theory, anti-PTCH/SHH chemotherapeutics for Nigerian ameloblastoma cases andalso infers the possible additional use of anti-p53 agents

АЛЛОГЕННЫЕ КОСТНОПЛАСТИЧЕСКИЕ МАТЕРИАЛЫ: СОВРЕМЕННОЕ СОСТОЯНИЕ ПРОБЛЕМЫ

Worldwide population aging and associated with it epidemics of osteoporosis, widespread of bone and joint reconstructive surgery and first of all joint replacement lead to explosive growth of interest in bone grafting.Although autografts are still the golden standard in bone regeneration, allogeneic bone substitutes have reached a state that allows for their application with satisfying clinical results. However, it has repeatedly been supposed that the different allogeneic materials underwent different purification processes, which modifies bone regeneration properties of these materials and also for different safety conditions. In the present publication, the treatment of the precursor tissue, the safety conditions, and the regenerative possibilities of C+TBA bone blocks based in preclinical and clinical data are described. Thus, it is described how the risks of infections and also immunological reactions becomes completely eliminated, while the special purification process allows for preservation of the native structure of the bone block. Both the in vitro studies and the clinical trials including histological follow-ups showed the optimal regeneration properties of these bone blocks. It has been shown that the allogeneic bone grafts have been integrated without causing inflammatory anomalies at the implantation site. Altogether, the allogeneic bone substitute material serves as an excellent basis for the formation of new bone. Finally, the combination of the allogeneic C+TBA bone blocks with different antibiotics is described. Interestingly, it is possible to combine the allogeneic bone substitute ether with antibiotics in the sense of prophylaxis and/or with bone marrow aspirate in order to accelerate bone remodeling.Старение населения планеты и ассоциированная с ним эпидемия остеопороза, а также широкое распространение реконструктивных операций на костях и суставах, в первую очередь эндопротезирования, привели к взрывному росту интереса к костной пластике.Несмотря на то, что аутотрансплантаты по-прежнему остаются «золотым стандартом» при замещении костных дефектов, аллогенные костнозамещающие материалы достигли такого уровня качества, который позволяет с успехом применять их в клинической практике. Неоднократно высказывались предположения, что разные способы обработки различных типов аллогенных материалов по-разному меняют их регенеративные свойства и характеристики безопасности. В статье описана технология обработки исходного материала, перечислены требования к безопасности аллокости, регенеративные возможности костных блоков C+TBA. Приводятся подтверждающие данные доклинических и клинических исследований. Технология C+TBA позволяет практически полностью исключить риск развития иммунологических реакций и передачи инфекции, а специальные этапы обработки позволяют сохранить естественную структуру костного блока. Клинические и in vitro исследования с гистологическим контролем на разных этапах показали оптимальные регенеративные характеристики таких костных блоков. Аллогенная кость интегрировалась, не вызывая локальных воспалительных реакций в месте трансплантации. В целом аллогенные костнозамещающие материалы являются отличной основой для формирования новой кости. В статье описаны комбинации аллогенных костных блоков C+TBA с различными антибиотиками с целью профилактики инфекции и/или с пунктатом костного мозга для стимуляции перестройки кости

Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-β pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues.; Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype

Tissue reaction to sealing materials: different view at biocompatibility

Abstract The biodegradability of root canal sealers in areas other than the root canal system is crucial to the overall success rate of endodontic treatment. The aim of the present study was to investigate, the cell and tissue reaction to GuttaFlow and AHPlus, both in vitro and in vivo. For the in vitro experiments the materials were incubated with Human Periodontal Ligament Fibroblasts and cell proliferation and cytotoxicity analyses were performed. Additional fluorescence-microscope stainings were carried out in order to visualize cell growth and morphology. For assessment of the tissue reaction to the materials a subcutaneous implantation model in Wistar rats was employed and the inflammatory response to the materials was visualized by means of general and specific histology after 6 weeks. Human gingival fibroblasts proliferation seemed to be dependent upon dental material and cultivation time. After an incubation period of 96 hrs AHPlus proved to be significantly (p < 0.002) more cytotoxic than GuttaFlow, as only a small number of fibroblasts survived on AHPlus. In vivo, GuttaFlow was surrounded by a fibrous capsule and no degradation took place, while AHPlus induced a well-vascularized granulation tissue in which the material was phagocyted by macrophages. The results of this study demonstrate that a potential cytotoxic effect of a sealing material may beneficial in order to have antibacterial properties and induce self degradation when accidentally extruded over the apical foramen.</p

On the Ultrastructure and Function of Rhogocytes from the Pond Snail Lymnaea stagnalis

Rhogocytes, also termed "pore cells", occur as solitary or clustered cells in the connective tissue of gastropod molluscs. Rhogocytes possess an enveloping lamina of extracellular matrix and enigmatic extracellular lacunae bridged by cytoplasmic bars that form 20 nm diaphragmatic slits likely to act as a molecular sieve. Recent papers highlight the embryogenesis and ultrastructure of these cells, and their role in heavy metal detoxification. Rhogocytes are the site of hemocyanin or hemoglobin biosynthesis in gastropods. Based on electron microscopy, we recently proposed a possible pathway of hemoglobin exocytosis through the slit apparatus, and provided molecular evidence of a common phylogenetic origin of molluscan rhogocytes, insect nephrocytes and vertebrate podocytes. However, the previously proposed secretion mode of the respiratory proteins into the hemolymph is still rather hypothetical, and the possible role of rhogocytes in detoxification requires additional data. Although our previous study on rhogocytes of the red-blooded (hemoglobin-containing) freshwater snail Biomphalaria glabrata provided much new information, a disadvantage was that the hemoglobin molecules were not unequivocally defined in the electron microscope. This made it difficult to trace the exocytosis pathway of this protein. Therefore, we have now performed a similar study on the rhogocytes of the blue-blooded (hemocyanin-containing) freshwater snail Lymnaea stagnalis. The intracellular hemocyanin could be identified in the electron microscope, either as individual molecules or as pseudo-crystalline arrays. Based on 3D-electron microscopy, and supplemented by in situ hybridization, immunocytochemistry and stress response experiments, we provide here additional details on the structure and hemocyanin biosynthesis of rhogocytes, and on their response in animals under cadmium and starvation stress. Moreover, we present an advanced model on the release of synthesized hemocyanin molecules through the slit apparatus into the hemolymph, and the uptake of much smaller particles such as cadmium ions from the hemolymph through the slit apparatus into the cytoplasm

3D-ultrastructure, functions and stress responses of gastropod (Biomphalaria glabrata) rhogocytes.

Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastropods and other molluscs, with great variation in their number, shape and size. They are enveloped by a lamina of extracellular matrix. Their most characteristic feature is the "slit apparatus", local invaginations of the plasma membrane bridged by cytoplasmic bars, forming slits of ca. 20 nm width. A slit diaphragm creates a molecular sieve with permeation holes of 20×20 nm. In blue-blooded gastropods, rhogocytes synthesize and secrete the respiratory protein hemocyanin, and it has been proposed-though not proven-that in the rare red-blooded snail species they might synthesize and secrete the hemoglobin. However, the cellular secretion pathway for respiratory proteins, and the functional role(s) of the enigmatic rhogocyte slit apparatus are still unclear. Additional functions for rhogocytes have been proposed, notably a role in protein uptake and degradation, and in heavy metal detoxification. Here we provide new structural and functional information on the rhogocytes of the red-blooded freshwater snail Biomphalaria glabrata. By in situ hybridization of mantle tissues, we prove that rhogocytes indeed synthesize hemoglobin. By electron tomography, the first three dimensional (3D) reconstructions of the slit apparatus are provided, showing detail of highly dense material in the cytoplasmic bars close to the slits. By immunogold labelling, we collected evidence that a major component of this material is actin. By genome databank mining, the complete sequence of a B. glabrata nephrin was obtained, and localized to the rhogocytes by immunofluorescence microscopy. The presence of both proteins fit the ultrastructure-based hypothesis that rhogocytes are related to mammalian podocytes and insect nephrocytes. Reactions of the rhogocytes to deprivation of food and cadmium toxification are also documented, and a possible secretion pathway of newly synthesized respiratory proteins through the slit apparatus is discussed

Allogeneic Bone Grafting Materials - Update of the Current Scientific Status

Worldwide population aging and associated with it epidemics of osteoporosis, widespread of bone and joint reconstructive surgery and first of all joint replacement lead to explosive growth of interest in bone grafting. Although autografts are still the golden standard in bone regeneration, allogeneic bone substitutes have reached a state that allows for their application with satisfying clinical results. However, it has repeatedly been supposed that the different allogeneic materials underwent different purification processes, which modifies bone regeneration properties of these materials and also for different safety conditions. In the present publication, the treatment of the precursor tissue, the safety conditions, and the regenerative possibilities of C+TBA bone blocks based in preclinical and clinical data are described. Thus, it is described how the risks of infections and also immunological reactions becomes completely eliminated, while the special purification process allows for preservation of the native structure of the bone block. Both the in vitro studies and the clinical trials including histological follow-ups showed the optimal regeneration properties of these bone blocks. It has been shown that the allogeneic bone grafts have been integrated without causing inflammatory anomalies at the implantation site. Altogether, the allogeneic bone substitute material serves as an excellent basis for the formation of new bone. Finally, the combination of the allogeneic C+TBA bone blocks with different antibiotics is described. Interestingly, it is possible to combine the allogeneic bone substitute ether with antibiotics in the sense of prophylaxis and/or with bone marrow aspirate in order to accelerate bone remodeling