Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Acellular human glans extracellular matrix as a scaffold for tissue engineering: in vitro cell support and biocompatibility

ABSTRACT Objectives: Diseases of the genitourinary tract can lead to significant damage. Current reconstructive techniques are limited by tissue availability and compatibility. This study aims to assess if the decellularized human glans can be used as a biomaterial for penile reconstruction. Materials and Methods: Samples of the glans matrices were descellularized. We evaluate the presence of collagen type I and III, and elastic fibers. Biocompatibility assays were performed to assess the cytotoxic and non-cytotoxic interactions between the acellular matrix and 3T3 cells. The matrices were seeded with mesenchymal stem cells and were assessed for viability and integration of these cells. Biomechanical tests in native tissue, descellularized matrix and seeded matrix were performed to characterize their biomechanical properties. Results: The tissue architecture of the decellularized matrix of human glans was preserved as well as the maintenance of the biomechanical and biological properties. The analyzes of glans seeded with mesenchymal stem cells revealed the integration of these cells to the matrices, and its viability during two weeks "in vitro". Conclusion: The decellularization process did not alter the biological and biomechanical characteristics of the human glans. When these matrices were seeded they were able to maintain the cells integrity and vitality.

Modeling pulmonary fibrosis by abnormal expression of telomerase/apoptosis/collagen V in experimental usual interstitial pneumonia

Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.

Study on nano-structured hydroxyapatite/zirconia stabilized yttria on healing of articular cartilage defect in rabbit

PURPOSE: Articular Cartilage has limited potential for self-repair and tissue engineering approaches attempt to repair articular cartilage by scaffolds. We hypothesized that the combined hydroxyapatite and zirconia stabilized yttria would enhance the quality of cartilage healing. METHODS: In ten New Zealand white rabbits bilateral full-thickness osteochondral defect, 4 mm in diameter and 3 mm depth, was created on the articular cartilage of the patellar groove of the distal femur. In group I the scaffold was implanted into the right stifle and the same defect was created in the left stifle without any transplant (group II). Specimens were harvested at 12 weeks after implantation, examined histologically for morphologic features, and stained immunohistochemically for type-II collagen. RESULTS: In group I the defect was filled with a white translucent cartilage tissue In contrast, the defects in the group II remained almost empty. In the group I, the defects were mostly filled with hyaline-like cartilage evidenced but defects in group II were filled with fibrous tissue with surface irregularities. Positive immunohistochemical staining of type-II collagen was observed in group I and it was absent in the control group. CONCLUSION: The hydroxyapatite/yttria stabilized zirconia scaffold would be an effective scaffold for cartilage tissue engineering.

Inducción de tolerancia oral en animales de experimentación a colágeno Tipo II purificado / Induction of oral tolerance in animales of experimentation to purified Type II collagen

Las enfermedades autoimunitarias constituyen hoy en día un problema de salud de relevancia, especialmenteporque son enfermedades crónicas severas que afectan considerablemente la calidad de vida de los pacientes yademás porque en la mayoría de los casos el tratamiento es solamente paliativo e insatisfactorio pues no modifica la evolución de la enfermedad. Un ejemplo bien conocido de este tipo de enfermedad es el de la ArtritisReumatoidea. En este sentido interesantes estudios handemostrado que la inducción de la tolerancia inmunológica por vía oral es efectiva en enfermedades autoinmunes humanas, constituyendo una alternativaventajosa, simple y barata de tratamiento. Dos mecanismos parecen estar involucrados: interacción, anivel de mucosas, del antígeno con células linfocitarias reguladoras (LTreg) que ejercerían un efecto supresorde la respuesta inmunitaria específica contra el antígeno ingerido11, o interacción de células dendríticas concélulas epiteliales que tiene deficiencia en moléculas coestimulatorias (CD80 y CD86), lo que conduciría a inducción de tolerancia.