Generation of a human control iPS cell line (ESi080-A) from a donor with no rheumatic diseases.

Here, we report the establishment of the human iPS cell line N1-FiPS4F#7 generated from skin cells of a patient with no rheumatic diseases, thus obtaining an appropriate control iPS cell line for researchers working in the field of rheumatic diseases. The reprogramming factors Oct4, Sox2, Klf4 and c-Myc were introduced using a non-integrating reprogramming strategy involving Sendai Virus.

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research.

The unavailability of sufficient numbers of human primary cells is a major roadblock for in vitro repair of bone and/or cartilage, and for performing disease modelling experiments. Immortalized mesenchymal stromal cells (iMSCs) may be employed as a research tool for avoiding these problems. The purpose of this review was to revise the available literature on the characteristics of the iMSC lines, paying special attention to the maintenance of the phenotype of the primary cells from which they were derived, and whether they are effectively useful for in vitro disease modeling and cell therapy purposes. This review was performed by searching on Web of Science, Scopus, and PubMed databases from 1 January 2015 to 30 September 2019. The keywords used were ALL = (mesenchymal AND ("cell line" OR immortal*) AND (cartilage OR chondrogenesis OR bone OR osteogenesis) AND human). Only original research studies in which a human iMSC line was employed for osteogenesis or chondrogenesis experiments were included. After describing the success of the immortalization protocol, we focused on the iMSCs maintenance of the parental phenotype and multipotency. According to the literature revised, it seems that the maintenance of these characteristics is not guaranteed by immortalization, and that careful selection and validation of clones with particular characteristics is necessary for taking advantage of the full potential of iMSC to be employed in bone and cartilage-related research.

Induced pluripotent stem cells for cartilage repair: current status and future perspectives.

The establishment of cartilage regenerative medicine is an important clinical issue, but the search for cell sources able to restore cartilage integrity proves to be challenging. Human mesenchymal stromal cells (MSCs) are prone to form epiphyseal or hypertrophic cartilage and have an age-related limited proliferation. On the other hand, it is difficult to obtain functional chondrocytes from human embryonic stem cells (ESCs). Moreover, the ethical issues associated with human ESCs are an additional disadvantage of using such cells. Since their discovery in 2006, induced pluripotent stems cells (iPSCs) have opened many gateways to regenerative medicine research, especially in cartilage tissue engineering therapies. iPSCs have the capacity to overcome limitations associated with current cell sources since large numbers of autologous cells can be derived from small starting populations. Moreover, problems associated with epiphyseal or hypertrophic-cartilage formation can be overcome using iPSCs. iPSCs emerge as a promising cell source for treating cartilage defects and have the potential to be used in the clinical field. For this purpose, robust protocols to induce chondrogenesis, both in vitro an in vivo, are required. This review summarises the recent progress in iPSC technology and its applications for cartilage repair.

In vitro repair model of focal articular cartilage defects in humans.

Articular cartilage lesions, which do not affect the integrity of subchondral bone, are not able to be repaired spontaneously, thus inducing cartilage degeneration and developing an arthrosic process. To avoid the need for prosthetic replacement, different cell treatments were developed with the aim of generating a repaired tissue with structure, biochemistry composition, and functional behavior equal or similar to those of natural articular cartilage.The following protocols describe the methods for harvesting articular cartilage explants both from pig and human specimens and isolating and culturing pig chondrocytes. Moreover, the methodology for an in vitro model of xenoimplant of pig chondrocytes in focal defects of human articular cartilage is described.

Differentiation of synovial CD-105(+) human mesenchymal stem cells into chondrocyte-like cells through spheroid formation.

Mesenchymal stem cells (MSCs) have the capacity to differentiate into several cell lineages, some of which can generate bone, cartilage, or adipose tissue. The presence of MSCs in the synovial membrane was recently reported. Data from comparative studies of MSCs derived from various mesenchymal tissues suggest that MSCs from synovial membranes have a superior chondrogenesis capacity. Previous chondrogenic differentiation studies have used the total population of MSCs, including cells with several MSC markers, such as CD44, CD90, CD105, or CD73. However the chondrogenic capacity of an individual population of MSCs has not been examined. Our aim was to study the chondrogenic capacity of the cellular MSC subset, CD105(+), derived from synovial membrane tissues of patients with osteoarthritis (OA) and normal donors. The tissues were digested with a cocktail of collagenase/dispase and the isolated MSCs were seeded into plates. The subpopulation of CD105(+)-MSCs was separated using a magnetic separator. The MSCs were then differentiated towards chondrocyte-like cells using a specific medium to promote spheroid formation. Spheroids were collected after 14, 28, and 46 days in chondrogenic medium and stained with hematoxylin, eosin, Safranin O or Alcian blue to evaluate the extracellular matrix. Immunohistochemistry was performed to study collagen types I (COLI) and II (COLII) and aggrecan expression. Phenotypic characterization of the isolated CD105(+)-MSCs shows that these cells are also positive for CD90 and CD44, but negatives for CD34 and CD45. In addition, this cellular subset expressed Sox-9. Spheroids appeared after 7 days in culture in the presence of chondrogenic medium. Our studies show no differences between MSCs obtained from OA and normal synovial membranes during chondrogenesis. The morphological analysis of spheroids revealed characteristics typical of chondrocyte cells. The intensity of Safranin O, Alcian blue and aggrecan staining was positive and constant throughout the culture period. However, the intensity of COL2 staining was higher at 28 days (84.29 +/- 0.1 U) than at 46 days (61.28 +/- 01 U), while COL1 staining was not detected in any samples analyzed. These results were confirmed by reverse transcriptase-polymerase chain reaction assays. We conclude that the cellular subset of CD105(+)-MSCs has chondrogenic capacity. The study also show the similar chondrogenic capacity of CD105(+)-MSCs cultured from normal and OA synovial membranes.

Mitochondrial activity is modulated by TNFalpha and IL-1beta in normal human chondrocyte cells.

OBJECTIVE: Pro-inflammatory cytokines play an important role in osteoarthritis (OA). In osteoarthritic cartilage, chondrocytes exhibit an alteration in mitochondrial activity. This study analyzes the effect of tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) on the mitochondrial activity of normal human chondrocytes. MATERIALS AND METHODS: Mitochondrial function was evaluated by analyzing the activities of respiratory chain enzyme complexes and citrate synthase, as well as by mitochondrial membrane potential (Deltapsim) and adenosine triphosphate (ATP) synthesis. Bcl-2 family mRNA expression and protein synthesis were analyzed by RNase protection assay (RPA) and Western-blot, respectively. Cell viability was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and apoptosis by 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) stain. Glycosaminoglycans were quantified in supernatant by a dimethyl-methylene blue binding assay. RESULTS: Compared to basal cells, stimulation with TNFalpha (10 ng/ml) and IL-1beta (5 ng/ml) for 48 h significantly decreased the activity of complex I (TNFalpha=35% and IL-1beta=35%) and the production of ATP (TNFalpha=18% and IL-1beta=19%). Both TNFalpha and IL-1beta caused a definitive time-dependent decrease in the red/green fluorescence ratio in chondrocytes, indicating depolarization of the mitochondria. Both cytokines induced mRNA expression and protein synthesis of the Bcl-2 family. Rotenone, an inhibitor of complex I, caused a significant reduction of the red/green ratio, but it did not reduce the viability of the chondrocytes. Rotenone also increased Bcl-2 mRNA expression and protein synthesis. Finally, rotenone as well as TNFalpha and IL-1beta, reduced the content of proteoglycans in the extracellular matrix of normal cartilage. CONCLUSION: These results show that both TNFalpha and IL-1beta regulate mitochondrial function in human articular chondrocytes. Furthermore, the inhibition of complex I by both cytokines could play a key role in cartilage degradation induced by TNFalpha and IL-1beta. These data could be important for understanding of the OA pathogenesis.

The osteoinductive potential of MTA (Mineral Trioxide Aggregate): a histologic study in rabbits

The objective of this study was to investigatebone response after implantation of MTA (MineralTrioxide Aggregate) in the rabbit mandible.Experiments were carried out on the rightmandibular body of 8 adult male rabbits. Theanimals were divided into two groups (controlgroup and test group). In this in vivo study, MTAwas used as an interpositional graft material incritical-size bone defects of rabbit mandibles.The animals were sacrificed on day 30 after surgery.The samples obtained from the mandibleswere subjected to histological procedures, whichpermitted the collection of sections with a thicknessof 60±10ìm. The sections were stained withHaematoxylin and Eosin and Goldner Trichromestain and examined under a light microscope.No important inflammatory reactions weredetected in any of the samples of the treatedgroup. The results confirm the excellent biocompatibilityof MTA. The implantation of MTA inbone defects led to bone regeneration 4 weeksafter surgery. However, the growth rate was notsignificant and the amount of newly formed bonewas limited with the use of MTA in this specificapplication. Sample examination did not suggestcomplete evidence of new bone growth from eitheran inductive or conductive perspective.The objective of this study was to investigatebone response after implantation of MTA (MineralTrioxide Aggregate) in the rabbit mandible.Experiments were carried out on the rightmandibular body of 8 adult male rabbits. Theanimals were divided into two groups (controlgroup and test group). In this in vivo study, MTAwas used as an interpositional graft material incritical-size bone defects of rabbit mandibles.The animals were sacrificed on day 30 after surgery.The samples obtained from the mandibleswere subjected to histological procedures, whichpermitted the collection of sections with a thicknessof 60+-10microm. The sections were stained withHaematoxylin and Eosin and Goldner Trichromestain and examined under a light microscope.No important inflammatory reactions weredetected in any of the samples of the treatedgroup. The results confirm the excellent biocompatibilityof MTA. The implantation of MTA inbone defects led to bone regeneration 4 weeksafter surgery. However, the growth rate was notsignificant and the amount of newly formed bonewas limited with the use of MTA in this specificapplication. Sample examination did not suggestcomplete evidence of new bone growth from eitheran inductive or conductive perspective

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Morphological study of root surfaces in teeth with adult periodontitis.

BACKGROUND: Our study correlates the histological alterations in the cementum (especially resorption areas) of teeth with the different stages of adult periodontitis. METHODS: Sixty-seven teeth affected by adult periodontitis and 7 healthy teeth extracted from patients over 40 years old were used. The teeth were divided into 3 groups according to radiographic data: group 1: five teeth with bone loss less than one-third of the normal alveolar height; group 2: thirty-one teeth with bone loss between one and two thirds; and group 3: thirty-one teeth with bone loss greater than two thirds. The samples were prepared for light and scanning electron microscopy, considering the gingival, middle, and apical thirds in each root. RESULTS: Two control teeth, 4 teeth in group 1, and all teeth in groups 2 and 3 showed resorption areas. Regarding the gingival third, the control teeth did not show any resorption, while 25% of affected teeth in group 1, 38.7% of teeth in group 2, and 35.5% of teeth in group 3 exhibited resorption. Regarding the middle third, 50% of affected teeth belonging to the control group and group 1; 67.7% of teeth in group 2; and 87.1% of teeth in group 3 showed resorption. Regarding the apical third, all teeth belonging to the control group and group 1 showed resorption, while 93.5% and 87.1% of teeth in groups 2 and 3, respectively, exhibited resorption. Most of the resorptions did not extend beyond the cementum. However, in 29.0% of teeth in group 2 and 38.7% of teeth in group 3, resorption had spread as far as the dentin. All the lesions in the control group and group 1 were practically repaired, while only 71.0% of teeth in group 2 and 61.3% of teeth in group 3 showed some sign of reparation. However, in groups 2 and 3, practically all lesions affecting dentin were repaired. CONCLUSIONS: These data suggest that the spread of root resorption is associated with inflammation. This study also suggests that the capacity for repair of root resorption is diminished with greater severity of periodontitis.