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The Influence of 2D and 3D Culture Systems on the Osteogenic Differentiation of hMSCs

Jacqueline Mimnaugh, RET Fellow 2011Science Teacher, Neuqua Valley High School

RET Mentor: Dr. Richard Gemeinhart Chicago Science Teacher Research (CSTR) Program – NSF-RET 2010

Experimental DesignIntroduction

Conclusion

Material and Methods

Results

Prof. A. Linninger, RET Program Director Dr. Richard Gemeinhart, Faculty Research Mentor Melanie Köllmer, Graduate Research Mentor Tracy Choung, RET and Fellow Researcher NSF Grant CBET-EEC-0743068 and the University of Illinois at Chicago

Acknowledgements

References1. Cowain, C. Evolving Concepts in Bone Tissue Engineering. Current Topics in

Developmental Biology, 66 (2005) 239 – 285.2. Pittenger, M.F., et al., Multilineage potential of adult human mesenchymal stem cells.

Science, 1999. 284(5411): p. 143-7.3. Zhang, S., Beyond the Petri dish. Nat Biotechnol, 2004. 22(2): p. 151-2.4. Abbott, A., Cell culture: biology's new dimension. Nature, 2003. 424(6951): p. 870-2.5. Burdick, J., Photoencapsulation of osteoblasts in injectable RGD-modified PEG

hydrogels for bone Tissue Engineering. Biomaterials 23 (2002) 4315–4323.

A variety of clinical reasons exist which require the replacement of bone tissue, including non-union fractures, craniofacial reconstruction, and trauma.1 The development of engineered bone tissue would provide a favorable option for individuals in need of bone tissue replacement.

Human mesenchymal stem cells (MSCs) have the ability to differentiate into a variety of cell types and are well suited for tissue engineering aplications.2 Traditional culturing of these cells has taken place on two dimensional plates; however, significant metabolic and morphological differences between cells cultured in 2D plates and cells in vivo have been reported. 3,4

As an alternative, the use of three dimensional scaffolds has been the subject of recent investigation. PEGDA scaffolds create a culturing environment that is more similar to the setting cells experience in vivo. 5

ObjectiveThe purpose of this study was to compare the osteogenic differentiation of hMSCs in a 2D culture system compared to a 3D culture system. Comparisons of osteogenic development indicators and mineralization were assessed over a four week period.

ProliferationWithin 3D PEGDA SPH scaffolds, hMSC viability peaked at day 14 and decreased at later time points suggesting a decrease in proliferation within the 3D system. In contrast, cells grown under 2D conditions showed a significant increase in proliferation at day 21 and remained at this level at day 28.

Project Overview:

1.Human mesenchymal stem cells were seeded into 2D and 3D culture systems.

2.Osteogenic differentiation medium was introduced after 24 hours and cells began to differentiate.

3.Osteoblasts were compared over 4 weeks for differences in:

• Proliferation• Differentiation Indicators• Mineralization

Figure 1. Gels were sliced into cylinders prior to seeding. Pores in the hydrogel range in size from 100 µm to 600µm.

The purpose of this study was to compare the osteogenic differentiation of hMSCs in a 2D culture system compared to a 3D culture system. Comparisons were based on three types of measurements: the amount of cell proliferation, the presence of proteins which indicate the differentiation of the cells into osteoblasts, and mineralization. Differences in cell viability occurred between the 2D and 3D systems. Proliferation in the SPH scaffolds peaked at day 14 and then decreased after that, whereas hMSCs in the 2D system increased in proliferation during the last two time points of the study. Differences in osteogenic differentiation indicators also occurred between the 2D and 3D systems. Alkaline phosphatase, an early indicator of osteogenic differentiation, peaked sooner in the 3D system than in the 2D system. Osteopontin, a mid to late indicator of osteogenesis, also reached its highest level sooner in the 3D system than in the 2D system. Mineralization was evident in both the 2D and 3D systems throughout the four week period as evidenced by both the Von Kossa stain images and calcium assay.

Creating the PEGDA Superporous HydrogelsA gas foaming technique was used to create the hydrogels. PEGDA, deionized water, foam stabilizer, radical initiator, and ammonium persulfate were added to a 4mL glass vial and heated. Citric acid and sodium bicarbonate were then added to create gas bubbles as the PEDGA polymerized. The gels were dried and sliced into cylinders 5 mm in diameter and 3 mm in height.

Seeding and Culturing the CellsMSCs were collected from two donors, one smoker and one non-smoker. The cells were seeded in equivalent densities, covered in basal medium, and incubated for 24 hours to allow adhesion. Basal medium was changed to osteogenic differentiation medium after 24 hours and replaced every 2 – 3 days.

Early IndicatorALP activity was significantly higher in the 3D scaffold compared to the 2D plate at all time points tested. In the 3D system, ALP activity reached its peak after 2 weeks of culture, while it took 4 weeks under 2D conditions. However, when normalized to cell number, ALP activity is higher under 2D conditions for 3 weeks of culture but increases at week four.

Mid/Late IndicatorWithin the 3D SPHs, osteopontin expression remained relatively constant over 4 weeks. Osteopontin expression levels are higher at day 2, 14 and 21 within the 3D culture system. In contrast, Osteopontin expression levels significantly increase at day 28 under 2D culture conditions compared to earlier time points and to 3D culture.

Mineralization Mineralization was present in both 2D and 3D culture systems. Dark spots indicate the presence of minerals. Images at 10X, Scale bar = 100µm

2D Polystyrene 48 Well Plate

3D Superporous Hydrogel

Mesenchymal Stem Cells

Osteoblast Osteoblast

Assays for Osteogenic Differentiation Alkaline Phosphatase Early Indicator Osteopontin Mid/Late Indicator Osteocalcin Late Indicator

Assays for Cell Proliferation MTS Cell Viability/Proliferation BCA Total Protein

Assays for Mineralization Calcium Calcium Production Von Kossa Staining Indicator of Mineralization

Assays for Cell Comparison

The following evaluations were made at days 2, 7, 14, 21 and 28: