Mesenchymal Stem Cells in Dense Collagen Scaffolds Promote Cortical Bone Healing
+1 Gao C; 1, 4Jiang F; 1Nguyen O; 3Serpooshan V; 1,4Julien M; 1, 4Wang H; 1, 4Li A; 3Nazhat S;
2, 4
Harvey EJ; 1,2,4 Henderson JE
Departments of 1Medicine, 2Surgery and 3Mining & Materials Engineering, McGill University, 4JTN Wong Laboratories, McGill University Health Centre,
Montreal, Quebec
[email protected]
INTRODUCTION
Poor bone quality is a common challenge in orthopaedic
reconstructive surgery and typically leads to complications such as nonunion in up to 25% of patients. The situation is exacerbated in the
elderly who have a decreased capacity for tissue regeneration and repair.
Interventions to promote bone regeneration in large skeletal defects
using allograft bone are largely ineffective due to its poor osteoinductive capability. The goal of this study was to determine if bone
marrow-derived mesenchymal stem cells (MSC) seeded in dense
collagen scaffolds could enhance bone healing in aged rodents.
osteoclast (TRAP) activity. In addition to more mineral, increased
staining for ALP and TRAP in the presence of cell-seeded scaffolds
(lower panels) suggested a higher rate of turnover.
E
D
LEFT
RIGHT
VK/TB
ALP
TRAP
METHODS
Ex vivo: MSC were isolated by adhesion to tissue culture plastic
from whole bone marrow harvested from the femurs and tibia of health
young adult donor mice. The cells were expanded in 2D culture ex vivo
before seeding at 6 x 104 in hydrated rat tail collagen to form 3D gels.
The collagen gels were then subjected to unconfined compression to
expel 98% of the fluid thus generating cell-seeded dense collagen
scaffolds (1). Metabolic activity of the MSC in osteogenic medium was
monitored on a weekly basis using Alamar Blue® and differentiation
was evaluated after 4 weeks using in situ staining with von
Kossa/toluidine blue (2).
In vivo: All live animal procedures were approved by a McGill
University animal use oversight committee. After 5 days of culture
MSC-seeded scaffolds, prepared as above, were transplanted into a 3mm
x 1mm full thickness defect drilled in the lateral aspect of the RIGHT
femur of 12 month old recipient mice. A similar defect in the LEFT
femur was either transplanted with an empty scaffold or left with no
intervention. Bone repair was evaluated after 4 or 6 weeks using
quantitative micro CT imaging and histological analyses. Bone mass and
architecture were defined by the parameters bone volume / tissue
volume (BV/TV), trabecular pattern factor (Tb.Pf) and trabecular
number (Tb.N). After CT imaging un-decalcified samples were
embedded at low temperature in MMA, sectioned at 5μm and stained for
mineral with Von Kossa/toluidine blue (VK/TB), alkaline phosphatase
(ALP) and tartrate resistance acid phosphatase (TRAP).
RESULTS
Ex Vivo: The dense collagen scaffold supported the survival of MSC
up to 4 weeks in culture (A). von Kossa staining of mineral deposited by
differentiated osteoblasts was seen in the cell-seeded scaffold (B) but not
in the empty scaffold (C) after 4 weeks of culture.
A
B
C
In Vivo: Quantitative micro CT analysis of bone healing in the femoral
defects at 4 weeks post operative showed a trend toward more bone in
the RIGHT defect, which received the cell-seeded scaffold, compared
with the LEFT defect that received an empty scaffold. The micro CT
data (D) was supported by histochemical staining (E) of 5μm sections of
plastic embedded bone for mineral (VK/TB), osteoblast (ALP) and
Quantitative micro CT analysis at 6 weeks post operative showed a
significant increase in bone regeneration in RIGHT femoral defects
containing scaffolds with 3 x 105 MSC compared with untreated LEFT
femoral defects.
Q mCT
LEFT
RIGHT
P value
BV/TV
6.1±3.9
10.1±4.1
< 0.05
TrPf
18.1±4.0
8.6±4.8
<0.01
TrN
0.6±0.4
1.1±0.5
<0.02
DISCUSSION
The data provides clear evidence that dense collagen scaffolds can
support the growth and differentiation of MSC ex vivo. When
transplanted in vivo the cell-seeded scaffolds enhanced bone
regeneration in a rodent model of fracture repair. The presence of TRAP
activity within the defect occupied by the MSC-seeded scaffold indicates
infiltration by catabolic cells, which was not evident in the case of the
empty scaffold. MSC-seeded dense collagen scaffolds thus show
promise as a potential substitute for bone grafts for bone reconstruction.
REFERENCES
1. V Serpooshan, M Julien, O Nguyen, H Wang, A Li, N Muja, J.E.
Henderson and S.N. Nazhat 2010 Reduced hydraulic permeability of
three dimensional collagen scaffolds attenuates gel contraction and
promotes the growth and differentiation of mesenchymal stem cells.
2010 Acta Biomater (In Press)
2. S. Richard, G. Valverde-Franco, G.A. Tremblay, T. Chen, N. Torabi,
G. Vogel, M. Morel, P Cleroux, S. Komarova, M.L. Tremblay, W. Li, A.
Li, Y- J Gao and J. E. Henderson. 2005 Ablation of the Sam68 RNAbinding protein protects mice from age-related bone loss. PLoS Genetics
v1, e74
ACKNOWLEDGEMENTS
Poster No. 1870 • ORS 2011 Annual Meeting