Sequential growth factor exposure of human Ad-MSCs improves chondrogenic differentiation in an osteochondral biphasic implant.

Joint cartilage damage affects 10-12% of the world's population. Medical treatments improve the short-term quality of life of affected individuals but lack a long-term effect due to injury progression into fibrocartilage. The use of mesenchymal stem cells (MSCs) is one of the most promising strategies for tissue regeneration due to their ability to be isolated, expanded and differentiated into metabolically active chondrocytes to achieve long-term restoration. For this purpose, human adipose-derived MSCs (Ad-MSCs) were isolated from lipectomy and grown in xeno-free conditions. To establish the best differentiation potential towards a stable chondrocyte phenotype, isolated Ad-MSCs were sequentially exposed to five differentiation schemes of growth factors in previously designed three-dimensional biphasic scaffolds with incorporation of a decellularized cartilage matrix as a bioactive ingredient, silk fibroin and bone matrix, to generate a system capable of being loaded with pre-differentiated Ad-MSCs, to be used as a clinical implant in cartilage lesions for tissue regeneration. Chondrogenic and osteogenic markers were analyzed by reverse transcription-quantitative PCR and cartilage matrix generation by histology techniques at different time points over 40 days. All groups had an increased expression of chondrogenic markers; however, the use of fibroblast growth factor 2 (10 ng/ml) followed by a combination of insulin-like growth factor 1 (100 ng/ml)/TGFß1 (10 ng/ml) and a final step of exposure to TGFß1 alone (10 ng/ml) resulted in the most optimal chondrogenic signature towards chondrocyte differentiation and the lowest levels of osteogenic expression, while maintaining stable collagen matrix deposition until day 33. This encourages their possible use in osteochondral lesions, with appropriate properties for use in clinical patients.

Maternal cafeteria diet exposure primes depression-like behavior in the offspring evoking lower brain volume related to changes in synaptic terminals and gliosis.

Maternal nutritional programming by caloric exposure during pregnancy and lactation results in long-term behavioral modification in the offspring. Here, we characterized the effect of maternal caloric exposure on synaptic and brain morphological organization and its effects on depression-like behavior susceptibility in rats' offspring. Female Wistar rats were exposed to chow or cafeteria (CAF) diet for 9 weeks (pre-pregnancy, pregnancy, and lactation) and then switched to chow diet after weaning. By postnatal day 60, the male Wistar rat offspring were tested for depressive-like behavior using operational conditioning, novelty suppressed feeding, sucrose preference, and open-field test. Brain macro and microstructural morphology were analyzed using magnetic resonance imaging deformation-based morphometry (DBM) and western blot, immunohistochemistry for NMDA and AMPA receptor, synaptophysin and myelin, respectively. We found that the offspring of mothers exposed to CAF diet displayed deficient motivation showing decrease in the operant conditioning, sucrose preference, and suppressed feeding test. Macrostructural DBM analysis showed reduction in the frontomesocorticolimbic circuit volume including the nucleus accumbens (NAc), hippocampus, and prefrontal cortex. Microstructural analysis revealed reduced synaptic terminals in hippocampus and NAc, whereas increased glial fibrillary acidic protein in hippocampus and lateral hypothalamus, as well as a decrease in the hippocampal cell number and myelin reduction in the dentate gyrus and hilus, respectively. Also, offspring exhibited increase of the GluR1 and GLUR2 subunits of AMPA receptor, whereas a decrease in the mGluR2 expression in hippocampus. Our findings reveal that maternal programming might prime depression-like behavior in the offspring by modulating macro and micro brain organization of the frontomesocorticolimbic circuit.

A Cellularized Biphasic Implant Based on a Bioactive Silk Fibroin Promotes Integration and Tissue Organization during Osteochondral Defect Repair in a Porcine Model.

In cartilage tissue engineering, biphasic scaffolds (BSs) have been designed not only to influence the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone, promoting the implant's integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a BS based on the assembly of a cartilage phase constituted by fibroin biofunctionalyzed with a bovine cartilage matrix, cellularized with differentiated autologous pre-chondrocytes and well attached to a bone phase (decellularized bovine bone) to promote cartilage regeneration in a model of joint damage in pigs. BSs were assembled by fibroin crystallization with methanol, and the mechanical features and histological architectures were evaluated. The scaffolds were cellularized and matured for 12 days, then implanted into an osteochondral defect in a porcine model (n = 4). Three treatments were applied per knee: Group I, monophasic cellular scaffold (single chondral phase); group II (BS), cellularized only in the chondral phase; and in order to study the influence of the cellularization of the bone phase, Group III was cellularized in chondral phases and a bone phase, with autologous osteoblasts being included. After 8 weeks of surgery, the integration and regeneration tissues were analyzed via a histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular BSs reached a Young's modulus of 805.01 kPa, similar to native cartilage. In vitro biological studies revealed the chondroinductive ability of the BSs, evidenced by an increase in sulfated glycosaminoglycans and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, in Group I, the defects were not reconstructed. In Groups II and III, a good integration of the implant with the surrounding tissue was observed. Defects in group II were fulfilled via hyaline cartilage and normal bone. Group III defects showed fibrous repair tissue. In conclusion, our findings demonstrated the efficacy of a biphasic and bioactive scaffold based on silk fibroin and cellularized only in the chondral phase, which entwined chondroinductive features and a biomechanical capability with an appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

A Bioactive Cartilage Graft of IGF1-Transduced Adipose Mesenchymal Stem Cells Embedded in an Alginate/Bovine Cartilage Matrix Tridimensional Scaffold.

Articular cartilage injuries remain as a therapeutic challenge due to the limited regeneration potential of this tissue. Cartilage engineering grafts combining chondrogenic cells, scaffold materials, and microenvironmental factors are emerging as promissory alternatives. The design of an adequate scaffold resembling the physicochemical features of natural cartilage and able to support chondrogenesis in the implants is a crucial topic to solve. This study reports the development of an implant constructed with IGF1-transduced adipose-derived mesenchymal stem cells (immunophenotypes: CD105+, CD90+, CD73+, CD14-, and CD34-) embedded in a scaffold composed of a mix of alginate/milled bovine decellularized knee material which was cultivated in vitro for 28 days (3CI). Histological analyses demonstrated the distribution into isogenous groups of chondrocytes surrounded by a de novo dense extracellular matrix with balanced proportions of collagens II and I and high amounts of sulfated proteoglycans which also evidenced adequate cell proliferation and differentiation. This graft also shoved mechanical properties resembling the natural knee cartilage. A modified Bern/O'Driscoll scale showed that the 3CI implants had a significantly higher score than the 2CI implants lacking cells transduced with IGF1 (16/18 vs. 14/18), representing high-quality engineering cartilage suitable for in vivo tests. This study suggests that this graft resembles several features of typical hyaline cartilage and will be promissory for preclinical studies for cartilage regeneration.

Thymidylate synthase gene variants as predictors of clinical response and toxicity to fluoropyrimidine-based chemotherapy for colorectal cancer.

BACKGROUND: Fluoropyrimidines form the chemotherapy backbone of advanced and metastatic colorectal cancer (CRC). These drugs are frequently associated with toxicity events that result in dose adjustments and even suspension of the treatment. The thymidylate synthase (TYMS) gene is a potential marker of response and toxicity to fluoropyirimidines as this enzyme is the molecular target of these drugs. Our aim was to assess the association between variants of TYMS with response and toxicity to fluoropyrimidines in patients with CRC in independent retrospective and prospective studies. METHODS: Variants namely rs45445694, rs183205964, rs2853542 and rs151264360 of TYMS were genotyped in 105 CRC patients and were evaluated to define their association with clinical response and toxicity to fluoropyrimidines. Additionally, the relationship between genotypes and tumor gene expression was analyzed by quantitative polymerase chain reaction. RESULTS: The 2R/2R (rs45445694) was associated with clinical response (p=0.05, odds ratio (OR)=3.45) and severe toxicity (p=0.0014, OR=5.21, from pooled data). Expression analysis in tumor tissues suggested a correlation between the 2R/2R genotype and low TYMS expression. CONCLUSIONS: The allele 2R (rs45445694) predicts severe toxicity and objective response in advanced CRC patients. In addition, the alleles G(rs2853542) and 6bp-(rs151264360) are independent predictors of response failure to chemotherapy. This is the first study made on a Latin American population that points out TYMS gene variants have predictive values for response and toxicity in patients with CRC treated with fluoropyrimidine-based chemotherapy.

Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries.

Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

Evaluation of in vivo pathogenicity of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis with different enzymatic profiles in a murine model of disseminated candidiasis.

Six isolates of the Candida parapsilosis complex with different enzymatic profiles were used to induce systemic infection in immunocompetent BALB/c mice. Fungal tissue burden was determined on days 2, 5, 10, and 15 post challenge. The highest fungal load irrespective of post-infection day was detected in the kidney, followed by the spleen, lung, and liver, with a tendency for the fungal burden to decrease by day 15 in all groups. Significant differences among the strains were not detected, suggesting that the three species of the "psilosis" group possess a similar pathogenic potential in disseminated candidiasis regardless of their enzymatic profiles.

Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer.

INTRODUCTION: Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-ß1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. METHODS: Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-ß1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. RESULTS: Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. CONCLUSION: Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair.

Las células TC-1 implantadas invaden a las fibras musculares esqueléticas adyacentes en un modelo murino de cáncer / Implanted TC-1 cells invade neighboring skeletal muscle fibers in a cancer murine model

La línea celular TC-1 se ha utilizado en la evaluación de la inmunoterapia antitumoral. No existen reportes sobre el efecto de las células TC-1 en tejidos adyacentes cuando se implantan en ratones C57BL/6. El objetivo de este trabajo fue evaluar la interacción entre las células TC-1 implantadas y las fibras musculares adyacentes. Se emplearon 8 ratones con células TC-1 implantadas y 3 ratones sin células. Se colectó el sitio del implante de las células tumorales a 10 días, las muestras se procesaron para microscopia de luz y electrónica de transmisión. Se realizaron tinciones con HyE y tricrómico de Masson, histoquímica con PAS, e inmunohistoquímica para identificar citoqueratinas, actina específica de músculo y metaloproteinasa de la matriz-9 (MMP-9). También se comparó el diámetro de las fibras musculares en ambos grupos de estudio. En el análisis histológico se observaron masas de células TC-1 que infiltran el tejido muscular y separan a las fibras entre sí. Las fibras musculares mostraron variaciones en la intensidad de la tinción y disminución de su diámetro. Se observaron masas de células tumorales TC-1 que invaden la fibra hacia el interior y logran atravesar la lámina externa y el sarcolema que las rodea. Se observó positividad a MMP-9 en el citoplasma de las células TC-1, y en el espacio entre las células tumorales y las fibras musculares. En el análisis ultraestructural se observó fragmentación y variaciones en el grosor de la lámina externa y vesículas subsarcolemales en el sitio en donde las células TC-1 invaden las fibras.Ahí, las células TC1 emiten proyecciones de membrana similares a pseudópodos....

TC-1 cells implanted in C57BL/6 mice are a model for evaluation of anti-tumor immunotherapy. To date there are no reports on the effect of implanted TC-1 cells upon neighboring striated muscle cells. The objective of this work was to evaluate the morphology of the interaction established among the implanted cells and the striated muscle cells. The study was carried out as follows: 8 adult C57BL/6 mice received 5x104 cells IP. As a control, 3 mice received no cells. 10 days after cells injection, no signs of tumor are present yet, and the site of cells injection was collected for morphological studies. Samples were processed for light and transmission electron microscopy. Histological sections were stained with H & E, Masson trichromic method, PAS histochemistry and immunohistochemistry for cytocheratins AE1/AE3, muscle specific actin and for matrix metalloproteinase-9. Cross section diameter of muscle sections was compared among experimental and control groups. The histological evaluation showed groups of tumor cells, infiltrating the spaces among muscle fibers. Muscle fibers showed variations in the cross section diameter as well as in the staining pattern. TC-1 cells were seen very close to muscle cells, invading the lamina externa and sarcolema to finally form groups of cells located within the sarcoplasm. This finding was demonstrated by the specific immunolabel for each kind of cell. Reactivity for metalloproteinase-9 was observed within the tumor cells and in the space mediating between the tumor cells and the muscle fiber. At the ultrastructural level, variations of the thickness of lamina externa were observed, as well as interruptions of this structure. Sarcolema also showed fragmentation, and close to these sites a number of subsarcolemmal vesicles were seen. In the vicinity of the muscle fiber, TC-1 cells formed membrane projections directed towards muscle membrane...

Decrease of virulence for BALB/c mice produced by continuous subculturing of Nocardia brasiliensis.

BACKGROUND: Subculturing has been extensively used to attenuate human pathogens. In this work we studied the effect of continuous subculturing of Nocardia brasiliensis HUJEG-1 on virulence in a murine model. METHODS: Nocardia brasiliensis HUJEG-1 was subcultured up to 130 times on brain heart infusion over four years. BALB/c mice were inoculated in the right foot pad with the bacteria subcultured 0, 40, 80, 100 and 130 times (T0, T40, T80 T100 and T130). The induction of resistance was tested by using T130 to inoculate a group of mice followed by challenge with T0 12 weeks later. Biopsies were taken from the newly infected foot-pad and immunostained with antibodies against CD4, CD8 and CD14 in order to analyze the in situ immunological changes. RESULTS: When using T40, T80 T100 and T130 as inoculums we observed lesions in 10, 5, 0 and 0 percent of the animals, respectively, at the end of 12 weeks. In contrast, their controls produced mycetoma in 80, 80, 70 and 60% of the inoculated animals. When studying the protection of T130, we observed a partial resistance to the infection. Immunostaining revealed an intense CD4+ lymphocytic and macrophage infiltrate in healing lesions. CONCLUSIONS: After 130 in vitro passages of N. brasiliensis HUJEG-1 a severe decrease in its virulence was observed. Immunization of BALB/c mice, with these attenuated cells, produced a state of partial resistance to infection with the non-subcultured isolate.

An experimental model of peripheral neuropathy induced in rats by Karwinskia humboldtiana (buckthorn) fruit.

Intoxication by Karwinskia humboldtiana (buckthorn) fruit presents a neurological picture similar to that of Guillain-Barré syndrome. In this report, we describe an experimental animal model of peripheral neuropathy induced by buckthorn fruit. Four groups of Wistar rats received one oral dose of 1.5 g/kg followed by oral doses of 0.5 g/kg at days 3, 7, 10, and 14 of dried and ground buckthorn fruit in aqueous suspension. Rats were sacrificed at 24, 48, 58, and 112 days after initial dose. Treated animals developed progressive paralysis through 58 days, then completely recovered by 112 days. Sciatic nerves showed segmental demyelination and cellular infiltrates until 58 days after exposure and then remyelinating changes at 112 days. This experimental model for peripheral neuropathy is reproducible and easy to handle. Its manipulation is relatively innocuous and allows us to study reversible peripheral nerve damage. This model can be developed in other animal species and may be useful to test new therapies for peripheral neuropathy.