Biomedical Engineering Reference
In-Depth Information
12.8
SEEDING BONE MARROW STEM CELLS ON NANO-APATITE
SCAFFOLDS
Besides hUCMSCs, human bone marrow mesenchymal stem cells (hBMSC) were also seeded on
CPC-based nano-apatite scaffolds. Adult mesenchymal (or stromal) stem cells derived from the bone
marrow are multipotent, able to differentiate into neural tissue, cartilage, bone, or fat
[5-10]
. hBMSCs
are emerging as an important tool to engineer bone tissues. hBMSCs can be harvested from patient's
bone marrow, expanded in culture, and combined with a scaffold carrier to deliver the cells to defec-
tive bone.
hBMSCs were cultured at 37°C with 5% CO
2
in low glucose DMEM (Gibco, Carlsbad, CA).
This media was supplemented with 10% FBS (Hyclone, Minneapolis, MN), 1% PS, 0.25% genta-
micin, and 0.25% fungizone. This media is referred to as the control media. The osteogenic media
consisted of the control media supplemented with 100 nM dexamethasone, 0.05 mM ascorbic acid,
and 10 mM β-glycerophosphate. At 90% confluence, cells were harvested by rinsing with 0.25%
trypsin, 0.03% EDTA solution, and incubated at room temperature until the cells detached. A live/
dead cell viability assay was performed to assess hBMSC viability. Cell viability and proliferation
were investigated at 1, 4, and 8 days
[53]
. The cells were observed using epifluorescence microscopy.
The percentage of live cells was defined as
P
LIVE
N
LIVE
/(
N
LIVE
N
DEAD
), where
N
LIVE
number of
live cells and
N
DEAD
number of dead cells, in the same image
[54]
.
Figure 12.9
shows hBMSCs on
CPC-based nano-apatite scaffolds
[55]
. Live cells formed an almost confluent monolayer and attained
a polygonal morphology consistent with osteogenic differentiation. There were few dead cells on all
materials at day 8. In (E), the percent of live cells at 4 days was similar for CPC at (87.8
5.1)% and
CPC-chitosan at (87.3
2.1)% (
P
0.1). At 8 days, the viability of cells increased to (90.5
1.3)%
for CPC-chitosan and (90.7
3.8)% for CPC control. At 8 days, there was no significant difference
in the percent of live cells between CPC, CPC-chitosan, and TCPS (
P
0.1).
Figure 12.10
shows hBMSC attachment and mineral formation on CPC-chitosan at (A and B) 4
days and (C and D) 8 days
[55]
. The hBMSCs had a polygonal morphology, typical for stem cells
undergoing osteogenic differentiation. Arrows in Figure 12.10(A) indicate globular mineral formation
and collagen bundles. In Figure 12.10(B), mineral formation is more clearly seen as granules. Similar
features and morphologies have been shown to be mineralization by cells in a previous study
[56]
.
The cell in Figure 12.19(C) is indicated by the letter “C” and the cytoplasmic extensions are indicated
by “E.” Mineralization on the cell surface at 8 days is shown in Figure 12.10(D).
Potential dental and craniofacial applications of the high-strength, nano-apatitic CPC-based
scaffold with the delivery of stem cells include major reconstructions of the maxilla or mandible
after trauma or tumor resection. These treatments would greatly benefit from a moldable implant
with shaping and aesthetic capabilities, possessing improved fracture resistance and rapid bone
regeneration. Other potential uses of the nano-apatitic composite scaffold include direct filling of
bulk bone defects, and minimally invasive surgeries such as
in situ
fracture fixation and percutane-
ous vertebroplasty to fill and strengthen osteoporotic bone lesions at risk for fracture. The strong
and tough CPC-based nano-apatitic composite scaffolds may be advantageous vehicles to deliver
stem cells to facilitate bone regeneration in a wide range of dental, craniofacial, and orthopedic
applications.
