Title: Human Mesenchymal Stem Cell Attachment and Spreading on 3D Porous Hydroxyapatite Bone Graft Substitu
1Human Mesenchymal Stem Cell Attachment and
Spreading on 3-D Porous Hydroxyapatite Bone Graft
Substitute
- Jorge I. Nuñez
- Department of Physics, UAB
- Department of Mechanical Engineering, UAB
- Mentor Susan L. Bellis, PhD, Department of
Physiology and Biophysics, UAB
2Background Implant Ideology
- Successful implant design is dependent on
understanding of implant surface interaction with
surroundings in vivo. - Challenge of engineering successful implant
implant needs to be strong while also being
biocompatible.
3Background Implant Ideology
- Metal and polymer coated implants being used are
strong and biocompatible but not effective at
promoting the adhesion of bone precursor cells. - This leads to formation of fibrous tissue layer
between implant and bone matrix which results in
eventual implant failure due to microfractures. - Coating implant surface with pro-adhesive
peptides improves recruitment of bone precursor
cells, mesenchymal stem cells, to implant surface.
4Mesenchymal Stem Cells
- Mesenchymal Stem Cells (MSCs), cells that reside
within the bone marrow, are pluripotent
progenitor cells. - MSCs have the ability to differentiate and
generate cartilage, muscle, tendon, ligaments,
fat, and bone. - In our case, MSCs differentiate along the
osteoblastic lineage to become bone forming
cells, i.e. osteoblasts.
5Pro-Adhesive Peptides and Cell Adhesion
- Pro-adhesive proteins, such as vitronectin and
fibronectin, contain and Arg-Gly-Asp (RGD)
sequence which binds to integrin receptors on the
cell surface. - Functionalizing metal / polymer implant surface
with RGD has been shown to increase cell adhesion
over biomaterial alone.
6MSC
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8- Hydroxyapatite (HA)
- Ca10(PO4)6(OH)2
- major constituent of bone mineral
- porous HA used as a scaffold for filling bone
defects - used as a coating for metal-based prosthetics
Calcium phosphates promote more rapid and
extensive bone formation in vivo than most other
biomaterials.
9Studies from our laboratory suggest that one of
the reasons HA is highly osseconductive is
because HA is superior to implant metals in
adsorbing blood proteins that promote MSC adhesion
10Should HA surfaces be functionalized with
adhesive peptides like RGD?
- Saturating concentrations of RGD could
potentially block the adsorption of serum
proteins that promote MSC adhesion and
differentiation.
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141.60
1.40
1.20
1.00
Cell Adhesion (fold to FBS)
0.80
0.60
0.40
0.20
0.00
1 mg/ml RGD FBS
10 mg/ml RGD FBS
100 mg/ml RGD FBS
1000 mg/ml RGD FBS
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16- Low RGD concentrations combined with pro-adhesive
serum proteins appears to promote cell adhesion
much better than RGD or FBS alone. - High RGD concentrations appears to inhibit cell
adhesion. - To simulate the environment in human body, we
coated HA disks with human serum.
17MSCs Attachment to RGD coated HA disks in
presence of FBS or HS
18MSCs Attachment and Spreading on RGD coated HA
disks
19- Trend of HA coated with RGD holds for human
serum. - I examined whether coating of 3-dimensional
porous HA scaffold with RGD would promote cell
adhesion.
20MSCs adhesion to Porous Scaffold
Cell Adhesion (Fold to FBS)
UNC
FBS
FBS/RGD
FBS/RGD
1 mg/ml
1000 mg/ml
21In vivo studies
- HA disks with different concentrations of RGD
coatings were implanted in rat tibiae - The implants were removed at 5 and 11 days and
sectioned. - The sections were stained with Hematoxylin and
Eosin.
22Bone formation in vivo
New Bone
Bone Marrow
HA disk
23Bone formation in vivo
New bone
Slide artifact
Osteoblasts lining disk surface
HA disk
24- In vivo bone formation was evaluated using two
parameters - Total amount of new bone formed.
- The amount of bone formed directly on the HA
surface.
25Bone formation in vivo
26Bone formation in vivo
27- Conclusion
- High RGD coatings appear to be detrimental to new
bone synthesis on HA biomaterials.
28Future
- Does RGD increase cell adhesion and bone
formation in vivo? - Do the trends seen with two samples hold over a
larger sample size? - How will the trend hold for RGD coated porous HA
in human serum or in vivo?
29Conclusion
- RGD peptides cooperate with serum proteins in
regulating MSCs adhesion. - Low RGD concentrations stimulate cell adhesion to
HA, suggesting potential therapeutic benefits. - High RGD concentrations inhibit cell adhesion to
HA, as well as in vivo bone formation. - Unlike metals or polymer biomaterials,
functionalizing calcium phosphates with high
concentration RGD is not beneficial.
30Acknowledgements
- Dr. Vohra and REU program.
- Dr. Bellis and Bellis lab group
- Amber
- Kristin
- Eric
- Faheem
- Pooja