High cell seeding densities of mesenchymal stem cells on gelatin scaffolds can improve cell proliferation during osteogenic differentiation. The study found that total RNA content, an indicator of cell proliferation, was significantly higher in osteogenic groups with 0.1M and 0.5M cell seeding densities compared to control groups. The 0.5M seeded osteogenic group also showed significantly higher RNA content than the 0.1M seeded osteogenic group, indicating that higher seeding densities can further improve proliferation. However, preliminary results showed that osteogenic gene expression, a measure of differentiation, did not differ between the 0.1M and 0.5M seeded osteogenic groups, suggesting that cell seeding density alone may not

1. Effect of Seeding Density of Mesenchymal Stem Cells on Cell Proliferation
and Osteogenic Differentiation Using Gelatin Scaffold
Jaideep Sahni, Zhongji Han, Shadi Othman
DEPARTMENT OF BIOLOGICAL SYSTEMS ENGINEERING, UNIVERSITY OF NEBRASKA-LINCOLN
INTRODUCTION
Total RNA Content
Total RNA content is an indirect indicator of cell proliferation.
The results showed that the RNA content of the osteogenic
groups [0.1 M (Figure 2A), 0.5 M (Figure 2B)] was
significantly higher than the control group (0.1 M , 0.5 M),
suggesting osteogenic differentiation could improve cell
proliferation; the results also showed that the RNA content of
the osteogenic group (0.5 M) was significantly higher than the
osteogenic group (0.1 M) (Figure 2C), suggesting that a high
seeding cell density could improve cell proliferation.
Gene Expression Analysis
Preliminary results showed that osteogenic differentiation
related gene expression was higher in osteogenic groups than in
the control group. There was no difference between the
osteogenic group (0.1 M) and the osteogenic group (0.5 M),
suggesting that the cell seeding density could not influence the
osteogenic differentiation.
RESULTS
1. KarageorgiouV, Kaplan D. Porosity of 3D biomaterial scaffoldsand
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
scaffoldfor osteoblasts.J Mater Sci Mater Med 2008;19:1173-1182.
BIBLIOGRAPHY
CONCLUSION
High cell seeding density could improve cell proliferation
during osteogenic differentiation.
Future Work
Collect more data for comparison of osteogenic differentiation
outcome between different cell seeding density group.
Fracture nonunion, osteoarthritis and other traumatic injuries of
underlying bone represent a major problem in regenerative medicine.
Autologous bone harvested from other anatomic locations and inserted
into 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. Bone
tissue engineering of autologous osteoprogenitor cells combined with
biodegradable scaffold has emerged as an alternative approach for the
treatment of large bone defect.
Porous gelatin scaffolds provide a three-dimensional structure for
attachment and proliferation of osteogenic cells by mimicking the
microenvironment of bones. Gelatin possesses these qualities and has a
high degree of biocompatibility, porosity and biodegradability. This
protein has been used before and shows great potential as a scaffold
(1).
Human mesenchymal stem cells (hMSCs) exhibit a high degree of
potency, ease of differentiation, purification and culturing, making it
one of the most common cell types used in tissue engineering (2).
Cell seeding density poses a more nuanced complication, as cell
density affecting differentiation via cell shape is a well-documented
phenomenon. Research suggests that osteogenic differentiation is
facilitated at low seeding densities, but little research has been done to
determine whether cell density is a stand-alone factor or whether it is
affected by other factors, such as scaffold type.
In this study, we collected week 3 samples to investigate osteogenic
differentiation of various seeding densities of hMSCs on gelatin
scaffold. 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).
MATERIALSAND METHODS
Preparation of Gelatin as an Engineered Bone Construct
Gelatin 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 a
20 ml syringe. They then were moved to 24-well plates after two hours. Medium was
changed every 3 days.
RNA Extraction
Fresh constructs were transferred into 1.5 ml centrifuge tubes. Samples were
homogenized in 1.5 ml Trizol (Life Technologies) using mortar plus liquid nitrogen
and RNA was extracted according to the single-step acid-phenol-guanidinium
method.
Real Time Reverse Transcription-Polymerase Chain Reaction
Osteogenic differentiation-related genes were quantified using Fast SYBR® Green
Master Mix. The transcript data were normalized to the housekeeping gene,
GAPDH. Expression of target genes was normalized to GAPDH and expressed as
the 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
TRANSLATIONALAND REGENERATIVE
MEDICINE IMAGING LABORATORY