Effect of Seeding Density of Mesenchymal Stem Cells on Cell Proliferation and Osteogenic Differentiation Using Gelatin Scaffold

Jaideep Sahni, Zhongji Han, Shadi OthmanDEPARTMENT OF BIOLOGICAL SYSTEMS ENGINEERING, UNIVERSITY OF NEBRASKA-LINCOLN

INTRODUCTION

Total RNA ContentTotal RNA content is an indirect indicator of cell proliferation.The results showed that the RNA content of the osteogenicgroups [0.1 M (Figure 2A), 0.5 M (Figure 2B)] wassignificantly higher than the control group (0.1 M , 0.5 M),suggesting osteogenic differentiation could improve cellproliferation; the results also showed that the RNA content ofthe osteogenic group (0.5 M) was significantly higher than theosteogenic group (0.1 M) (Figure 2C), suggesting that a highseeding cell density could improve cell proliferation.

Gene Expression AnalysisPreliminary results showed that osteogenic differentiationrelated gene expression was higher in osteogenic groups than inthe control group. There was no difference between theosteogenic group (0.1 M) and the osteogenic group (0.5 M),suggesting that the cell seeding density could not influence theosteogenic differentiation.

RESULTS

1. Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005;26:5474-5491.

2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 1999;284:143-147.

3. Rohanizadeh R, Swain MV, Mason RS. Gelatin sponges (Gelfoam) as a scaffold for osteoblasts. J Mater Sci Mater Med 2008;19:1173-1182.

BIBLIOGRAPHY

CONCLUSION

High cell seeding density could improve cell proliferationduring osteogenic differentiation.

Future WorkCollect more data for comparison of osteogenic differentiationoutcome between different cell seeding density group.

Fracture nonunion, osteoarthritis and other traumatic injuries ofunderlying bone represent a major problem in regenerative medicine.Autologous bone harvested from other anatomic locations and insertedinto the defect site is a representative therapy. However, in many cases,this approach suffers from additional pain, donor site morbidity,limited availability and longer time for patients in the hospital. Bonetissue engineering of autologous osteoprogenitor cells combined withbiodegradable scaffold has emerged as an alternative approach for thetreatment of large bone defect.

Porous gelatin scaffolds provide a three-dimensional structure forattachment and proliferation of osteogenic cells by mimicking themicroenvironment of bones. Gelatin possesses these qualities and has ahigh degree of biocompatibility, porosity and biodegradability. Thisprotein has been used before and shows great potential as a scaffold(1).

Human mesenchymal stem cells (hMSCs) exhibit a high degree ofpotency, ease of differentiation, purification and culturing, making itone of the most common cell types used in tissue engineering (2).

Cell seeding density poses a more nuanced complication, as celldensity affecting differentiation via cell shape is a well-documentedphenomenon. Research suggests that osteogenic differentiation isfacilitated at low seeding densities, but little research has been done todetermine whether cell density is a stand-alone factor or whether it isaffected by other factors, such as scaffold type.

In this study, we collected week 3 samples to investigate osteogenicdifferentiation of various seeding densities of hMSCs on gelatinscaffold. We also measured the total RNA content for each group.

Figure 2: Total RNA Content of Different Groups. (A) osteogenic differentiation media increased cell proliferation with 0.1 M cell seeding, (B) osteogenic differentiation media increased cell proliferation with 0.5 M cell seeding, (C) 0.5 M seeded groups showed more cell proliferation than the 0.1 M seeded groups, (**p<0.01, ***p<0.001).

MATERIALS AND METHODS

Preparation of Gelatin as an Engineered Bone ConstructGelatin constructs were seeded in 50 µl DMEM medium at 0.1 x 106 cells/scaffold(0.1 M) and 0.5 x 106 cells/scaffold (0.5 M) with the assistance of a vacuum using a20 ml syringe. They then were moved to 24-well plates after two hours. Medium waschanged every 3 days.

RNA ExtractionFresh constructs were transferred into 1.5 ml centrifuge tubes. Samples werehomogenized in 1.5 ml Trizol (Life Technologies) using mortar plus liquid nitrogenand RNA was extracted according to the single-step acid-phenol-guanidiniummethod.

Real Time Reverse Transcription-Polymerase Chain ReactionOsteogenic differentiation-related genes were quantified using Fast SYBR® GreenMaster Mix. The transcript data were normalized to the housekeeping gene,GAPDH. Expression of target genes was normalized to GAPDH and expressed asthe fold ratio relative to the control group using the 2-ΔΔCT method.

Figure 1: Scanning Electron Microscope Micrographs of Gelatin Scaffold (Gelfoam®). (A) gelatin sponge showing a porous structure, (B) at higher magnification closed pores and the flat walls between the pores (black arrow) were observed in gelatin sponge. (3)

A B

C

TRANSLATIONAL AND REGENERATIVE MEDICINE IMAGING LABORATORY