(485az) Effect of Initial Seeding Density On Hydroxyapatite-Chitosan-Gelatin (HCG) 3-D Scaffold Development

Developing biomimetic materials has become an important approach to induce specific cellular outcomes in tissue regeneration. To promote bone tissue regeneration from human mesenchymal stem cells (hMSCs), our lab has developed a three dimensional (3-D) biomimetic scaffold by incorporating hydroxyapatite, chitosan, and gelatin (HCG) into a porous matrix and has demonstrated its potency on hMSC growth, retention, and osteogenic differentiation in static culture (Zhao et al, 2002, 2006). In addition to the biomimetic construct environment, the development of functional 3-D constructs also requires uniform spatial cellular distribution and growth throughout the scaffold. Although these factors have been extensively studied in other scaffolds, little research has been done to examine how initial seeding density and distribution may influence long term hMSC growth patterns, characteristics, and differentiation in the HCG scaffold (Ding et al, Zhao et al 2006). In addition, hydroxyapatite, chitosan, and gelatin are all bioactive materials and how the absorption or adherence of proteins and growth factors to these substrates affects cell growth and construct development has not yet been studied (Hoven et al, Sharpe et al). In this study, we aim to examine the effects of initial cell seeding density and media preincubation on hMSC growth and osteogenic differentiation in the HCG scaffold. The pore size of the fabricated HCG scaffold was found to be 93 ± 27 μm using SEM while seeding efficiency using depth filtration was 30.5% ± 11.0%. SEM images indicate cells adhere primarily to the surface of the scaffold upon seeding but ingrowth into the scaffold can be seen after 21 days of culture. Preliminary growth studies indicate scaffold preincubation and serum protein absorption influence initial scaffold growth greatly with a 62% decrease in cell number after 21 days growth in scaffolds without media preincubation. Preincubated scaffolds, however, increased in cell number by 79% from the initial seeding value after only 7 days of growth and showed a 4.3 fold increase after 21 days of culture. The preliminary results indicate pre-incubation of our HCG scaffold affects cell growth characteristics by decreasing the lag phase of cell growth in 3-D culture. Growth data is currently being collected to elucidate the affect of initial seeding density on long term (21 day) culture as well osteogenic differentiation as measured by alkaline phosphatase expression, calcium deposition and gene expression. Together, the results will improve construct fabrication methods and enhance osteogenic potential.

References:

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