1. A critical-sized defect in bone represents a clinical challenge that
necessitates the use of a bone regeneration strategy. Platelet-rich
plasma (PRP) provides an easily accessible, patient-derived complex
mixture of growth factors and cytokines. In early studies, PRP was
applied as a platelet gel for bone healing. However, the mechanical
integrity and degradation characteristics of platelet gels were
problematic for bone regeneration. Recently, PRP has been
incorporated into various gels, sponges, and electrospun scaffolds by
first converting PRP into a bioactive lyophilized powder termed
preparations rich in growth factors (PRGF). Including PRGF within a
support scaffold allows for localized, sustained delivery of the
platelet-derived factors. Previous in vitro studies using various bone
cell models have shown that cell proliferation is increased by PRP
treatment, but effects on osteoblast differentiation and function have
been variable. Investigating effects of PRGF, rather than PRP, can
provide a more controlled approach for evaluating how cells in the
osteoblast lineage respond to the complex mixture of platelet-derived
factors present in PRP.
Study Objective: To determine the response of MG-63 osteoblast-
like cells in 2-D culture to medium supplemented with PRGF.
Lyophilized Platelet-Rich Plasma Increases Osteoblast Proliferation and Alkaline Phosphatase Activity
Rachel Rone, Scott Sell, and Natasha Case
Department of Biomedical Engineering, Parks College of Engineering, Aviation and Technology, Saint Louis University, Saint Louis, MO
Introduction
Materials and Methods
Discussion and Conclusion
Results
• MG-63 human osteosarcoma cell line (ATCC® CRL-1427™) was
used as an osteoblast model.
• PRGF powder was created from human PRP that was isolated by
standard techniques, subjected to freeze-thaw cycles to lyse platelets,
and then lyophilized.
• Cells were expanded and seeded (~10,000 cells/cm2 ) in high-glucose
DMEM with 5% EquaFETAL bovine serum (Atlas Biologicals) and
1% penicillin/streptomycin.
• Three days after seeding, cell layers were rinsed in serum-free
medium and then cultured in medium containing 1% serum ± 0.5
mg/mL PRGF for 6 days, with a medium change on day 3.
• For analysis, cell layers were scraped in lysis buffer (100 mM Tris-
HCL, 1 mM MgCl2, 0.1% Triton X) and sonicated.
• As a measure of osteoblast function , cell lysates were analyzed for
alkaline phosphatase (ALP) activity based upon conversion of the
substrate P-nitrophenyl phosphate.
• Cell lysates were also analyzed for total protein content (BCA assay).
0
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1% Serum 1% serum + PRGF
Protein(mg)
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8000
1% Serum 1% Serum + PRGF
ALPActivity
(nmol/min/mgprotein)
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160
ITS ITS + PRGF
Protein(mg)
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7000
ITS ITS + PRGF
ALPActivity
(nmol/min/mgprotein)
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Day 0 Day 3 Day 6
Protein(mg)
• Experiments were conducted in low-serum medium to reduce
interfering effects of bovine serum on the response to PRGF.
• MG-63 cells survived and proliferated in medium with 1%
serum.
• PRGF enhancement of MG-63 protein content was consistent
with previous PRP studies in bone cells that showed increased
cell proliferation with PRGF addition.
• The increase in ALP activity by PRGF treatment, observed in
both low-serum and ITS+-supplemented medium, indicated
that one important aspect of osteoblast function was enhanced
by platelet-derived factors.
• Follow-up studies will evaluate PRGF effects on other aspects
of osteoblast function.
• Future experiments will evaluate the response of MG-63 cells
seeded onto 3-D scaffolds in which PRGF is incorporated.
CONCLUSION: PRGF, a lyophilized form of PRP, supports
increased proliferation and alkaline phosphatase activity in MG-
63 osteoblast-like cells under low serum conditions.
0
1000
2000
3000
4000
5000
6000
1% Serum 1% Serum + PRGF
ALPActivity
(nmol/min/mgprotein)
0
50
100
150
200
250
1% Serum 1% Serum + PRGF
Protein(mg)
Figure 4. PRGF effect on ALP activity
(experiment B). Significant increase
with 6 days of PRGF treatment (p<0.01).
Figure 5. PRGF effect on protein
content (experiment B). Significant
increase with PRGF (p<0.05).
Acknowledgements
• Technical assistance from K. Hixon, A. Dunn, and A. Martin
• Funding provided by the Parks College of Engineering,
Aviation and Technology
Figure 6. PRGF effect on ALP activity
(ITS+ experiment). 1% serum was
replaced with a serum-free supplement
containing insulin (ITS+). Significant
increase with PRGF (p<0.001) at 6 days.
Figure 7. PRGF effect on ALP activity
(ITS+ experiment). The amount of
protein was not different with PRGF
addition to medium with ITS+.
Figure 1. Establishing a low-serum culture
model for testing effects of PRGF. Protein
content was increased with culture time,
indicating proliferation in medium with 1% serum.
Figure 2. PRGF effect on ALP activity
(experiment A). The addition of PRGF
significantly increased (p<0.001) ALP
activity in the cell layer at 6 days.
Figure 3. PRGF effect on protein content
(experiment A). The addition of PRGF also
significantly increased (p<0.001) the
amount of protein in the cell layer.
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