Seeding Osteoblastic Cells into a Macroporous Biodegradable CaP/PLGA Scaffold by a Centrifugal Force

This study aims to construct a hybrid biomaterial by seeding osteoblastic cells into a CaP/PLGA scaffold by a centrifugal force. Constructs are evaluated with respect to potential application in bone tissue engineering. Cells adher, spread, and form a layer of tissue lining the scaffold and are capable of migrating, proliferating, and producing mineralized matrix. We have demonstrated that the centrifugal force is highly efficient for constructing a hybrid biomaterial, which acts similarly to bone explants in a cell culture environment. In this way, these constructs could mimic an autogenous bone graft in clinical circumstances. Such a strategy may be useful for bone tissue engineering.

Modelling oxygen diffusion and cell growth in a porous, vascularising scaffold for soft tissue engineering applications

Soft tissue engineering presents significant challenges compared to other tissue engineering disciplines such as bone, cartilage or skin engineering. The very high cell density in most soft tissues, often combined with large implant dimensions, means that the supply of oxygen is a critical factor in the success or failure of a soft tissue scaffold. A model is presented for oxygen diffusion in a 15-60 mm diameter dome-shaped scaffold fed by a blood vessel loop at its base. This model incorporates simple models for vascular growth, cell migration and the effect of cell density on the effective oxygen diffusivity. The model shows that the dynamic, homogeneous cell seeding method often employed in small-scale applications is not applicable in the case of larger scale scaffolds such as these. Instead, we propose the implantation of a small biopsy of tissue close to a blood supply within the scaffold as a technique more likely to be successful. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

The adult human intervertebral disc (IVD) is normally avascular. Changes to the extracellular matrix in degenerative disc disease may promote vascularisation and subsequently alter cell nutrition and disc homeostasis. This study examines the influence of cell density and the presence of glucose and serum on the proliferation and survival of IVD cells in 3D culture. Bovine nucleus pulposus (NP) cells were seeded at a range of cell densities (1.25 × 10(5)-10(6) cells/mL) and cultured in alginate beads under standard culture conditions (with 3.15 g/L glucose and 10 % serum), or without glucose and/or 20% serum. Cell proliferation, apoptosis and cell senescence were examined after 8 days in culture. Under standard culture conditions, NP cell proliferation and cluster formation was inversely related to cell seeding density, whilst the number of apoptotic cells and enucleated "ghost" cells was positively correlated to cell seeding density. Increasing serum levels from 10% to 20% was associated with increased cluster size and also an increased prevalence of apoptotic cells within clusters. Omitting glucose produced even larger clusters and also more apoptotic and senescent cells. These studies demonstrate that NP cell growth and survival are influenced both by cell density and the availability of serum or nutrients, such as glucose. The observation of clustered, senescent, apoptotic or "ghost" cells in vitro suggests that environmental factors may influence the formation of these phenotypes that have been previously reported in vivo. Hence this study has implications for both our understanding of degenerative disc disease and also cell-based therapy using cells cultured in vitro.

Improved Bioprocess with CHO-hTSH Cells on Higher Microcarrier Concentration Provides Higher Overall Biomass and Productivity for rhTSH

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (A degrees C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 A degrees C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 x 10(6) cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (similar to 80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (similar to 9 x 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce similar to 1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Bioinspired polyethersulfone-based hollow fiber membranes as the scaffolds in renal assist device for protein-bound toxins removal from blood

Dissertation for obtaining the Master degree in Membrane Engineering

Artificial thymus: a tissue engineering strategy

The thymus is the central organ responsible for the generation of T lymphocytes (1). Various diseases cause the thymus to produce in- sufficient T cells, which can lead to immune-suppression (2). Since T cells are essential for the protection against pathogens, it is crucial to promote de novo differentiation of T cells on diseased individuals. The available clinical solutions are: 1) one protocol involving the transplant of thymic stroma from unrelated children only applicable for athymic children (3); 2) for patients with severe peripheral T cell depletion and reduced thymic activity, the administration of stimu- lating molecules stimulating the activity of the endogenous thymus (4). A scaffold (CellFoam) was suggested to support thymus regen- eration in vivo (5), although this research was discontinued. Herein, we propose an innovative strategy to generate a bioartificial thymus. We use a polycaprolactone nanofiber mesh (PCL-NFM) seeded and cultured with human thymic epithelial cells (hTECs). The cells were obtained from infant thymus collected during pediatric cardio-tho- racic surgeries. We report new data on the isolation and characterization of those cells and their interaction with PCL-NFM, by expanding hTECs into relevant numbers and by optimizing cell seeding methods.

Mesenchymal stem cells culture on poly(vinylidene fluoride) piezoelectric microspheres

Dissertação de mestrado em Biofísica e Bionanossistemas

Compressed collagen gel: a novel scaffold for human bladder cells.

Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo-tissue level. This controlled, cell-independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell-seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation.

A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration.

Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen-poly(lactic acid-co-ɛ-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen-PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen-PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.

L’étude de l’interaction entre les chondrocytes et le collagène modifié par le 4-Hydroxynonénal : implication dans le développement de l’arthrose

OBJECTIF: Récemment, nous avons démontré que la modification du collagène type II (Col II) par le 4-hydroxynonénal (HNE), un produit de la peroxydation lipidique, est augmentée dans le cartilage arthrosique sans qu’on sache la signification de cette augmentation dans la pathogenèse de l’arthrose. L’objectif de cette étude vise à démontrer que cette modification affecte l’interaction chondrocytes/matrice extracellulaire (MEC) et en conséquence induit des changements phénotypiques et fonctionnels de ces cellules. METHODES: Des plaques de culture ont été préalablement cotées avec du Col II puis traitées avec du HNE (0.1-2 mM) excepté le puits contrôle. Les chondrocytes ont été ensuite ensemencés puis incubés pendant 48 heures. La viabilité des cellules est évaluée par le test MTT. Le Western blot est utilisé pour mesurer l’expression des molécules d’adhésion (l’ICAM-1 et l’intégrine α1β1), de la cyclooxygenase-2 (COX-2), du Col II ainsi que la phosphorylation de la p38 MAPK, ERK1/2 et NF-κB-p65. La RT-PCR en temps réel est utilisée pour mesurer l’expression de l’ARNm de l’ICAM-1, des intégrines α1β1, de la COX-2 et de la métalloprotéinases-13 (MMP-13). La détermination de l’expression de l’ICAM-1 à la surface des cellules est réalisée par cytométrie de flux. Des kits commerciaux ont servi pour mesurer le niveau de la MMP-13, de la prostaglandine E2 (PGE2), de l’activité de la caspase-8 et de la phosphorylation de la p38 MAPK, ERK1/2 et NF-κB-p65. RESULTATS: La modification du Col II par 0.2 mM HNE induit significativement l’expression des molécules d’adhésion telles que l’ICAM-1 et l’intégrine α1β1, de la MMP-13 sans avoir un effet sur la morphologie, la survie et le phénotype cellulaires. Nos résultats montrent aussi une forte augmentation de la phosphorylation de la p38 MAPK, d’ERK1/2 et de NF-κB-p65. Cependant, la modification du Col II par 2 mM HNE affecte la morphologie et la viabilité cellulaires et induit l’activité de la caspase-8. Elle inhibe fortement l’expression des integrines α1β1 et du Col II ainsi que la phosphorylation de l’ERK1/2 et de NF-κB-p65, mais par contre, induit significativement la production de la COX-2 et son produit la PGE2 ainsi que la phosphorylation de la p38 MAPK. Fait intéressant, le prétraitement des complexes HNE/Col II par 0.1 mM de carnosine empêche les changements phénotypiques et fonctionnels des chondrocytes. CONCLUSION : Ces nouveaux résultats suggèrent le rôle important de la modification du Col II par le HNE dans l’arthrose, en affectant le phénotype et le fonctionnement cellulaires des chondrocytes. La carnosine, par sa capacité de neutraliser le HNE, a révélé d’être un agent promoteur dans le traitement de l’arthrose.

Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degreesC. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degreesC in 5% CO2 atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable too] in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied. (C) 2004 Elsevier B.V. All rights reserved.

Resposta de fibroblastos da gengiva de pacientes jovens e idosos e efeito da laserterapia de baixa potência e de fator de crescimento sobre estas células

Pós-graduação em Reabilitação Oral - FOAR

General Protocol for the Culture of Cells on Plasma-Coated Electrospun Scaffolds

As opposed to culture on standard tissue-treated plastic, cell culture on three-dimensional scaffolds impedes additional challenges with respect to substrate preparation, cell seeding, culture maintenance, and analysis. We herewith present a general route for the culture of primary cells, differentiated cells, or stem cells on plasma-coated, electrospun scaffolds. We describe a method to prepare and fix the scaffolds in culture wells and discuss a convenient method for cell seeding and subsequent analysis by scanning electron microscopy or immunohistology.

Vascular engineering for bypass surgery

Since the introduction of synthetic vascular grafts in the 1960s, only two-stage endothelial cell seeding has demonstrated any significant improvement over conventional vascular grafts, and its benefits have yet to be demonstrated on a large scale. Tissue engineering is a rapidly expanding field with great potential, but efforts to construct tissue-engineered arterial grafts have, to date, yielded little clinical success. This review explores the latest approaches to the construction of a superior vascular graft, along with its potential for use in the clinic in the future.