Cell Seeding

Topics: Scanning electron microscope, Electron microscope, Cell culture Pages: 9 (2725 words) Published: December 1, 2008
Seeding cells into needled felt scaffolds for tissue engineering applications

SUMMARY:

Tissue engineering methods are under development that will enable the repair or replacement of a variety of tissues, including articular cartilage and bone. To engineer functional tissue it is necessary that scaffolds initially be seeded with a large number of cells distributed evenly throughout the scaffold structure. It previously has been shown that, compared to static seeding conditions, seeding scaffolds under dynamic conditions facilitates high seeding densities and even distributions of cells. The efficiency of seeding HOSTE85 cells and bovine chondrocytes into needled felt scaffolds following agitation at different speeds was determined. Seeding efficiency was determined using the Hoechst 33258 assay, and cell viability was assessed using the Alamar Blue™ assay. The distribution of cells within the scaffolds was imaged using scanning electron microscopy. It was found that the optimum seeding conditions varied for HOSTE85 cells and bovine chondrocytes, with different agitation speeds leading to different seeding efficiencies, cell viabilities, and distributions of cells within scaffolds. The optimum agitation speeds for seeding a high number of viable cells into scaffolds so that they were arranged evenly were 300 rpm for HOSTE85 cells and 200 rpm for bovine chondrocytes. This proposal highlights the studies of improved seeding of cells into scaffolds using dynamic rather than static seeding methods. [1,2] The optimum seeding conditions for the two cell types were different, as shown by the efficiency with which cells were seeded into the scaffolds and the arrangement of cells within the scaffolds. It is unknown why the cells respond differently to agitation; however, it is postulated that it may be due to differences in cell size or density. A study of this nature therefore is vital prior to commencing tissue engineering work in order to determine the optimum parameters for seeding scaffolds. HYPOTHESIS: The studies of improved seeding of cells into scaffolds using dynamic rather than static seeding methods.

SPECIFIC AIMS:

Musculoskeletal injuries affect one in seven Americans, causing chronic pain, severely reducing the person’s quality of life, and costing the nation approximately $254 billion per year.[3] Damage to musculoskeletal tissues, such as articular cartilage and bone, may be the result of trauma, for example sports or road traffic accidents, or it may occur as a result of a disease, such as osteoarthritis.[4] It has been estimated that in excess of 40 million people in the United States suffer with arthritis each year.[5] Tissue engineering methods are being developed that will allow the repair or replacement of such diseased or damaged tissues. Currently, many approaches for the engineering of organs and tissues are under development. One strategy is to take a biopsy of tissue, isolate the cells by enzymatic digestion, and then expand them in culture. The cells are then seeded into a suitable scaffold structure that will support the proliferating cells. The cell seeded construct must then be cultured under appropriate conditions to allow extracellular matrix formation and tissue regeneration.[1] There are many important parameters that must be optimized to enable the formation of functional tissues. It is important that the scaffold employed supports cell attachment, extracellular matrix and tissue formation, and that the constructs initially be seeded with a high number of cells that are distributed evenly throughout the entire scaffold.6 For example, when tissue engineering articular cartilage, a high seeding density is necessary to prevent fibrous tissue formation.[6] It also is necessary for cells to be distributed evenly throughout the scaffold structure for normal tissue formation.[1]

It previously has been reported that higher seeding densities and more uniform...

References: 1. Li Y, Ma T, Kniss DA, Lasky LC, Yang ST. Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices. Biotechnol Prog 2001;17:935– 944.
2. Vacanti CA, Vacanti JP. Bone and cartilage reconstruction. In: Lanza R, Langer R, Vacanti J, editors. Principles of tissue engineering. London, UK: Academic; 1997. p 619–631.
3. American Association of Orthopaedic Surgeons Orthopaedic Fast Facts. Available at http://orthoinfo.aaos.org/fact/thr_report.cfm?Thread_ID_93&topcategory_About%200rthopaedics.
4. Buckwalter JA, Mankin HJ. Articular cartilage. II. Degeneration and osteoarthrosis, repair, regeneration, and transplantation. J Bone Joint Surg Am 1997;79A:612–632.
5. Center for Disease Control and Prevention. Targeting arthritis: Thenation’s leading cause of disability. At-A-Glance, 2001. Available at http://www.cdc.gov/nccdphp/aag/aag_arthritis.htm.
6. Freed LE, Vunjak-Novakovic G. Culture of organized cell communities. Adv Drug Del Rev 1998;33:15–30.
7. Robinson G, Gray T. Electron microscopy. II. Practical procedures. In: Bancroft J, Stevens A, editors. Theory and practice of histological techniques. 4th ed. New York: Churchill Livingstone; 1996. p 585–625
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