Biomaterial-Mediated Control over Macrophage Behavior in Bone Regeneration
Pamela L. Graney1, Seyed-Iman Roohani-Esfahani2, Hala Zreiqat2, Kara L. Spiller1
1Biomaterials
& Regenerative Medicine Laboratory, Department of Biomedical Engineering, Drexel University, Philadelphia, PA, USA
2Biomaterials and Tissue Engineering Research Unit, The University of Sydney, NSW, Australia
Scope
COMPARISON OF DIRECT AND INDIRECT MACROPHAGE – SCAFFOLD INTERACTIONS
Methods
SIGNIFICANCE
MACROPHAGE – SCAFFOLD INTERACTIONS
M1 Markers
•  Approximately 6 million bone fractures occur annually in the U.S.1
•  5-10% fail to heal adequately1
•  Bone loss, failed fixation, infection, poor vascularization
•  Standard treatments involve harvesting tissue from other locations within the body or transplanting
donor tissue into the defect
•  Limited tissue supply, risk of rejection, donor site morbidity
•  Although small fractures heal perfectly, without scarring2, large bone defects remain a challenge3.
THERE
IS AN UNMET NEED FOR
TISSUE ENGINEERING
STRATEGIES THAT
UTILIZE SYNTHETIC MATERIALS TO HARNESS THE NATURAL ABILITY OF BONE TO REPAIR ITSELF
MACROPHAGES: CRUCIAL REGULATORS OF HEALING
Spectrum of Macrophage Populations
Macrophage Populations in Normal Healing
Classically Activated [M1]
QUANTITATIVE ANALYSIS OF GENE EXPRESSION
RNA isolated using TRIzol and RNeasy Mini Kit (Qiagen), and treated with DNase I (Invitrogen)
cDNA synthesis performed using High Capacity Reverse Transcription Kit (Applied Biosystems)
qRT-PCR performed using 20 ng cDNA per reaction & SYBR® Green PCR Master Mix (Applied Biosystems)
Expression of target genes was normalized to the reference gene, GAPDH
•  Data shown represent mean fold change over M0 ± standard error mean (n ≥ 4)
•  Statistical analysis completed in GraphPad Prism 6.0
•  ANOVA with Tukey’s post-hoc analysis test
•
•
•
•
Wound-healing
[M2a]
Regulatory
[M2c]
M2a Markers
Image modified from [4]
Desired Macrophage Response in Bone Repair
Results & Discussion
M2a Marker
TIME-DEPENDENT ANALYSIS OF DIRECT MACROPHAGE – SCAFFOLD INTERACTIONS
TCP-HA
Baghdadite
M2c Marker
Sr-HT Gahnite
1
0.1
p < 0.05
0.01
0.001
Day 1.5
Day 6
CCR7
1
0.1
Fold Change over GAPDH
IDEAL BONE SCAFFOLDS SHOULD MIMIC THE NATURAL HEALING RESPONSE:
STIMULATE M1 AT EARLY STAGES OF HEALING, PROMOTE M2 AT LATER STAGES
TNF-α
Fold Change over GAPDH
Fold Change over GAPDH
M1 Markers
M1 Markers
p < 0.05
0.01
0.001
0.0001
Day 1.5
Day 6
IL1B
1
0.1
p < 0.05
p < 0.05
0.01
0.001
Day 1.5
Day 6
•  Indirect interactions are not significantly different from the control.
•  Baghdadite and Sr-HT Gahnite scaffolds directly modulate macrophages responses.
NOVEL CERAMIC-BASED SCAFFOLDS
•  Recently, Roohani and Zreiqat engineered novel ceramic-based scaffolds, Baghdadite and Sr-HT
Gahnite, and demonstrated that both scaffolds, at high porosity (85%) and interconnectivity (100%),
exhibit enhanced ability to regenerate large bone defects under load, compared to clinically utilized
tricalcium phosphate-hydroxyapatite (TCP-HA) scaffolds.5,6
Baghdadite
Ca3ZrSi2O9
Strontium-Hardystonite (Sr-HT) Gahnite Sr-Ca2ZnSi2O7-ZnAl2O4
(TCP-HA)
Β-Ca3(PO4)2-Ca10(PO4)6(OH)2
In this work, we evaluated the behavior of primary human monocyte-derived macrophages on
novel ceramic scaffolds in vitro in terms of gene expression for a panel of markers indicative of
the M1, M2a and M2c phenotypes. Interestingly, while TCP-HA scaffolds induced chronic
inflammation, Baghdadite scaffolds promoted an M1-to-M2c transition, consistent with the M1to-M2 transition observed in normal healing. Our findings indicate that part of the success of
these scaffolds may be due to modulation of macrophage behavior by direct contact with
macrophages. We are currently confirming these results at the level of protein secretion and
investigating the use of these scaffolds as a platform to study M2c behavior in the absence of
IL-10.
TCP-HA
Composition
M2c Marker
Representative SEM Image
of ceramic-based scaffolds
WE HYPOTHESIZED THAT INTERACTIONS WITH MACROPHAGES CONTRIBUTE
TO THE SUCCESS OF THESE SCAFFOLDS TO PROMOTE TISSUE REGENERATION
Sr-HT Gahnite
Scaffold Name
M2a Markers
Images from [6]
Objective
The objective of this work is to understand the interactions between cells of the inflammatory
response and ceramic scaffolds proven to result in bone regeneration in animal models. Conclusion
•  Baghdadite scaffolds induce M1-like behavior at early times and promote a transition to M2c-like behavior at
later times, consistent with the natural M1-to-M2 transition in normal healing.
IMPROVED UNDERSTANDING OF THE INTERACTIONS BETWEEN SCAFFOLDS AND CELLS OF THE
INFLAMMATORY RESPONSE WILL AID IN THE DESIGN OF BIOMATERIALS
TO FACILITATE BONE REPAIR AND TISSUE REGENERATION.
REFERENCES
W HAT
ARE
THE
DIFFERENCES
BETWEEN
THESE
SCAFFOLDS
THAT
LEAD
TO
THIS
BEHAVIOR ?
[1] C. Cheung, Clin Podiatr Med Surg, 2005, 22, 631-641vii; [2] R.A. Carano, E.H. Filvaroff, Drug Discov Today,
2003, 8, 980-989; [3] M.R. Hausman, B.D. Rinker, Am J Surg, 2004, 187, 44S-55S; [4] D.M. Mosser, J.P. Edwards,
Nat Rev Immunol, 2008, 8, 958-969; [5] S.I. Roohani-Esfahani et al. Acta Biomaterialia, 2012, 8, 4162-4172; [6] S.I.
Roohani-Esfahani et al., Acta Biomaterialia, 2013, 9, 7014-7024.