The Ability For Self Regeneration Biology Essay

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chondrocyte, articular cartilage lacks the ability for self-regeneration. The regeneration of functional hyaline cartilage, using expanded chondrocytes and biodegradable polymers, is an important research area in the field of cell-based therapies and tissue engineering that may provide a solution for cartilage reconstruction. Tissue engineering plays an increasingly important role in the functional repair of diseased and missing cartilage that complements reconstructive and orthopedic surgery. A common remedial approach is to harvest cells from a small tissue biopsy from the donor, expand them in vitro, and seed the

population into biodegradable and porous scaffolds, producing an enhanced semiartificial structure for cell distribution and population in culture. The cell-cultivated construct is then implanted in vivo for

extracellular matrix production and, eventually, for cartilage regeneration. It has been asserted that a high-quality cell/scaffold construct requires high spatial uniformity of seeded cells, high scaffold cellularity to

enhance the tissue development rate, and sufficient nutrient and oxygen supplies to maintain cell viability. In producing high-quality cell-assisted implantable constructs, cell density may significantly affect cartilage and bone formation. Primary isolated chondrocytes from a small biopsy specimen, which may itself be diseased, hinder the in vitro expansion of a clinically useful number of chondrocytes and they have the tendency to de-differentiate in ex vivo culture. Uniform cell distribution and proliferation

in engineered scaffolds are also critical issues in regenerative medicine. The primary goal of research in cell seeding and cultivation is to promote cell population and uniformity in scaffolds before they are implanted in vivo. Production of uniform high cell density is limited, though, by the complexity of the

scaffold assembly, insufficient migratory abilities of cells into the structure during seeding, and the tendency of cell movement to the periphery and out of the implantable materials in culture. Consequently, prolonged in vitro cell culture has been required to meet effectual cell density and distribution profiles prior to implantation. Unfortunately, long in vitro culture periods may induce cellular dedifferentiation, limiting the utility of the procedure for patients requiring tissue therapy under expedited terms and yet increasing the possibility of in vitro contamination leading to infection in vivo.To circumvent elongated cell-culture requirements and improve cell uniformity, a number of methods to increase cell-seeding density have been investigated. A cell-seeding device, utilizing the synergistic effects of vacuum, centrifugal force, and fluid flow, has been used with porous scaffolds. Magnetic nanoparticles have been used to guide fibroblast cells even through commercially available scaffolds; the magnetite nanoparticles were directed with a magnetic field to induce ‘‘mag-seeding,â€Â subsequently increasing cell density and seeding efficiency. Seeding cells on scaffolds in bioreactors to enhance cell density and uniformity has also been investigated. A more simplified method was pursued to increase seeded cell density using protein gels to temporarily engage a well-distributed cell mixture with porous scaffolds. Various gels prepared as cellencapsulating scaffolds have been investigated for high cell-density seeding. Here, we present a simple gelation method using alginate gel, a natural polymer, as gelling material to incorporate a high cell density in 3D porous scaffolds during cell seeding and restrain the cells in the scaffolds from escaping during the subsequent cell culture, aimed to achieve ultimate high cell density and uniformity in porous structures. Alginate gels have been studied for cartilage tissue engineering applications as a matrix for cellular encapsulation and culture. The method involves no complex equipment, and the scaffold system is entirely constructed from natural polymers that have proven their biocompatibility and are favored for a wide spectrum of tissue engineering applications. The aim of this study was to investigate and to compare by flow cytometric methods, morphological changes, cellular viability and apoptosis of human chondrocytes cultured in gel-assisted and conventional non-gel-assisted cell-seeding methods for cartilage tissue engineering applications. Hyaluronidase, trypsin, collagenase from Clostridium histoliticum, alginic acid sodium salt from brown algae, CaCl2, NaCl and calcein-AM were from Sigma-Aldrich (St. Louis, MO, US. Osteoarthritic articular chondrocytes were isolated using Green et al., 1971 and Kuettner et al. 1982 protocols from patients with osteoarthritis undergoing arthroplasty under sterile techniques (CF 2 Hospital, Bucharest, Romania. The obtained pellet was divided into three equal parts (the one for classical monolayer culture and the others for alginate microencapsulation and control samples). The cells were seeded into 1.5 cm chambers (Nalge Nunc International, Naperville, I L, USA) which provide enough surface area to allow 4x104 isolated chondrocytes to proliferate in DMEM medium. The cultures were maintained at 370 C in a humidified 5 % CO2 for 7 days. The chondrocytes (4x104) were suspended in 5 ml NaCl 155mM and mixed whit 0.06 g sodium alginate. The alginate/cell suspension is formed into droplets of uniform size and shape obtained by forcing the suspension through the small orifice of a needle and then breaking up the stream into droplets. The

droplets fall into 70 ml calcium chloride solution 102 mM. In presence of calcium chloride the sodium alginate polymerizes and forms microcapsules in which the chondrocytes are entrapped. The obtained

microcapsules (10-12 microcapsules/ml culture medium) were transferred into 1.5 cm chambers in 1 ml DMEM medium, and cultured for 7 days at 370 C in a humidified 5 % CO2. The microcapsules were removed from DMEM medium and washed twice with PBS buffer. The gelled alginate support was liquefied using a chelating agent to remove the calcium ions from the gel by treating with 10 ml mixture of Tris-HCl 0.01 M, 60 mM sodium citrate and 0.2M NaCl. Then the cells were centrifugated, washed with PBS and analyzed by flow cytometry. Analysis of the scattered light by flow cytometry in the mode FSC/SSC provides information about cell size and structure. After 7 days of chondrocytes culture (seeding) using a standard protocol, we noticed the separation of the same regions as before, with maintenance of roughly the same percentage of viable cells (24.5%). However, when we grew the chondrocytes in alginate microspheres culture, the cells presented a more homogeneous distribution in the FSC/SSC plots, and about 64% of them remained in the region (R1) of the dot-plot, described as viable region. Consequently, we can conclude that the three-dimensional culture system in alginate microspheres allows chondrocyte division, while normal cell morphology is retained in a single homogeneous population. We compared the viability of chondrocytes grown in standard culture or with alginate microspheres. When the cultures started, viable osteoarthritic chondrocytes represented 43% of the population, with a MFI of 1452. After 7 days of culture using standard method, viable chondrocytes amounted to 54% of the cell population, and had a reduced MFI of 987. In the same time, chondrocytes grown in mixture with alginate microspheres were 98% viable, with an MFI of 1146, which is closer to the initial value. In this regard, it is important to mention that we have previously demonstrated that the loss of esterase activity was an early event that occurred before phosphatidylserine exposure. On the basis of these results we can conclude that by including the chondrocytes in alginate microspheres, in a threedimensional culture system, cellular viability is significantly improved, as shown by both viable cell percentage and esterase activity measurements. The osteoarthritic chondrocytes before seeding, as well as from monostat or alginate microsphere cultures, were analyzed by flow cytometry for externalization of

phosphatidylserine (PS, labeled with annexin-V) and for cellular membrane permeability (staining with propidium iodide). Fig. 3 shows a region-based comparative analysis by which we simultaneously identified viable cells, as well as apoptotic and necrotic cells, using a double staining with Annexin-V-FITC (FL1) and propidium iodide (FL2). The initial osteoarthritic chondrocytes had 38% viable cells, 15% cells in early apoptosis, and 32% cells in late apoptosis (necrosis), respectively. After 7 days of growth in standard culture, the population was composed of 39% viable cells, 29% early apoptotic cells, and 31% late apoptotic (necrotic) cells. When the cells were grown in alginate microspheres, we found 73% viable cells and only 24% apoptotic cells after 7 days of culture. We were thus able to identify a better correlation with the established FSC/SSC analysis for apoptosis. This analysis allowed distinction between two populations: viable cells (high FSC and low SSC, region R1), and apoptotic cells (low FSC and high SSC, region R2). Our data, in the context of other published findings, underline the advantages of culturing chondrocytes with alginate microspheres. In fact, chondrocytes cultured using this method represent a single phenotypic population with a tight cellular uniformity as shown by the FSC/SSC analyses and show a very high viability (over 90%). These findings recommend the implementation of this biotechnology in tissue engineering. The results we obtained also emphasize the advantages of using flow cytometry to quantitatively evaluate various methods of growing chondrocytes in tissue culture. This analysis method was very useful in characterizing cell viability, which may represent the starting point for using thismethod to screen for growth factors and better culture conditions. 1527

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