Macrophage Dynamics of the Foreign Body Response to Biomaterials and Wound Healing
Tony Yu, Sina Nassiri, Kara Spiller
School of Biomedical Engineering, Science, and Health Systems Drexel University, Philadelphia, PA, USA
Introduction
• Study of macrophage dynamics in wound healing in two experiments: Foreign body
response and chronic wounds
• Biomaterials and implantable medical devices – part of the solution to many unmet
clinical needs
• Chronic wounds – impairs normal wound healing via deletion of M1->M2 switch [1]
• Macrophage dysfunction in both biomaterial implantation due to the foreign body
response (FBR) and in chronic wounds
Figure 1: Biomaterials particularly sensitive to the foreign body response. A. Glial scarring around
implanted microelectrodes block the conductance of neuronal activity. B. Vascularization is blocked
by fibrous capsule. C. The fibrous capsule acts as a diffusion barrier in drug delivery devices.
[1] Nassiri, Sina. J Invest Dermatol 2015
Characterization
of Gelatin Hydrogels
< Figure 4: Properties of crosslinked
hydrogels. A) Young’s modulus determined
in unconfined compression. B) Crosslinking
density, determined from equilibrium
swelling theory. C) Equilbrium swelling
ratio. D) Change in mass over time,
normalized to starting mass. # indicates
statistical significance among all groups at
these time points (* denotes p<0.05, one
way ANOVA performed at each time point).
** denotes p<0.01, *** denotes p<0.005,
**** denotes p<0.001
iNOS (M1 Macrophages)
e
• M1– “Classically activated” inflammatory macrophages [2]
• M2a– “Alternatively activated” pro-healing macrophages
• M2c– Remodeling macrophages (not included in this study)
Figure 3: Normal wound healing: the inflammatory response with a peak in the
M1 macrophages at the early stage, followed by the pro-healing response or the
accumulation of M2 macrophages at the later stage.
Masson’s Trichrome
[3] Spiller, Kara. Biomaterials 2014
System of Equations
Schematic of Wound Healing
< Figure 8: Schematic of macrophage
dynamics in normal wound healing. It is
assumed that there is an immediate
recruitment of the M0 macrophage to the
wound site upon injury. The M0 macrophage
can either polarize to M1, polarized to M2, or
undergo apoptosis leaving the system. The M1
macrophage either transitions to the M2 or
undergo apoptosis. The M2 macrophage can
proliferate or undergo apoptosis.
𝑑𝑀0
= 𝑅 + −𝑘1 ∗ 𝑀0 + −𝑘2 ∗ 𝑀0 − (𝑑0 ∗ 𝑀0)
𝑑𝑡
𝑑𝑀1
= 𝑘1 ∗ 𝑀0 + −𝑘3 ∗ 𝑀1 − (𝑑1 ∗ 𝑀1)
𝑑𝑡
𝑑𝑀2
= 𝑘2 ∗ 𝑀0 + 𝑘3 ∗ 𝑀1 + (𝑘4 ∗ 𝑀2) − (𝑑2 ∗ 𝑀2)
𝑑𝑡
Initial Conditions
Cells (based on
in vitro experiments)
@t=0
M0
M1
M2
1 x 106
0
0
List of Parameters
Math Model Assumptions
1)
2)
Arg1 (M2 Macrophages)
3)
4)
5)
6)
7)
Modeling of recruited, not resident macrophages
Monocytes are recruited to the wound site immediately
after t > 0
All recruited monocytes differentiate into macrophages
Once polarized, M1 and M2 macrophages cannot
differentiate back to M0
M2 macrophages cannot transition back to M1
Only M2 macrophage can proliferate
Rate constants calculated at specific time intervals are
constant over time
Math Model Results
A) Normal Wound Healing
There is a strong correlation between the amount of M2 macrophages and the thickness of the fibrous capsule
• Regulation of macrophages is key to successful wound healing where macrophage dysfunction is an issue
• M2 macrophages are responsible for formation of the fibrous capsule around biomaterials
• The math model described the macrophage dynamics in normal and chronic wounds using in vitro experiments
• Also described potential treatments for chronic wound done by other studies
In vitro experiments for mathematical model [3]
• Human monocytes - isolated using Ficoll and
Percoll density gradient centrifugation.
• Cell viability and performed flow
cytometry of CCR7 (M1 marker) and CD206 (M2
marker) expression
• Rate constants calculated from survival expression
• Survival Expression = % viable cells x expression
of marker from flow cytometry
[2] Spiller, Kara. Biomaterials. 2014
Figure 7 >: IHC stainting of Arg1 (a-i). H
denotes the hydrogel, while FC denotes the
fibrous capsule. Yellow boxes and insets
indicate the region of intensity
quantification. (j) Negative control (delete
primary) and (k) positive controls (spleen).
Scale bar is 200µm. (l) Quantification of
intensity of staining, determined from the
regions in the yellow boxes indicated on
each figure and quantified in ImageJ.
Statistical significance was determined using
two way ANOVA and Tukey’s post-hoc
analysis. (**p<0.01).
Conclusion
FBR of gelatin hydrogel implantation
• Gelatin hydrogels crosslinked in 0.05%, 0.1%, and 0.3%
glutaraldehyde concentrations.
• Characterized by mechanical stiffness, swelling,
crosslinking density, and degradation.
• Subcutaneously implanted in C57BL mice for 3, 10 and
21 days.
• Explanted at each time point for Masson’s Trichrome and
IHC staining of iNOS (M1 marker) and Arg1 (M2 marker)
Figure 2: Macrophages exist on a spectrum of phenotypes: M1, M2a, and M2c.
Figure 5 >: Masson’s Trichrome: Fibrous
capsule quantification (a-i). Black lines
indicate the thickness of the fibrous capsule.
(j) Representative image showing the
thickness of the fibrous capsule on the
muscle-facing (j) and skin-facing (k) sides of
the hydrogel. Scale bar is 200um. (l)
Statistical significance was determined using
two way ANOVA and Tukey’s post-hoc
analysis. ** denotes p<0.01, **** denotes
p<0.001.
< Figure 6: IHC staining of iNOS (a-i). H
denotes the hydrogel, while FC denotes the
fibrous capsule. Yellow boxes and insets
indicate the region of intensity
quantification. (j) Negative control (delete
primary) and (k) positive controls (spleen).
Scale bar is 200µm. (l) Quantification of
intensity of staining, determined from the
regions in the yellow boxes indicated on
each figure and quantified in ImageJ.
Statistical significance was determined using
two way ANOVA and Tukey’s post-hoc
analysis. (*p<0.05).
Methods
Macrophage Biology
B) Chronic Wound
Parameters
Description (rates)
Value (1/day)
Source (Survival
Expression)
d0
M0 death
0.201
Day 0 to day 3
d1
M1 death
0.465
Day 1 to day 3
d2
M2 death
0.611
Day 1 to day 3
k1
M0 polarization to M1
0.608
Day 0 to day 1
k2
M0 polarization to M2
0.0836
Day 0 to day 1
k3
M1 transition to M2
0.36
Day 3 to day 6
k4
M2 proliferation
0.405
Day 3 to day 6
C) Biomaterial
Implantation
D) Anti-inflammatory
drug @ day 0
E) Anti-inflammatory
drug @ day 3
Figure 9: A) The M0, M1, and M2 macrophage dynamics in the normal wound healing model. an initial infiltration of M1 macrophages representing the inflammatory response, followed by an accumulation of
the M2 macrophages representing the pro-healing response. B) The macrophage profile of a chronic wound with the deletion of M1->M2 terms and R= 0.1. C) Macrophage behavior in a foreign body response
to biomaterial implantation with R = 0.5. D) A treatment to a chronic wound (k3 = 0, R = 0.1) with k1 = 0 representing the delivery of anti-inflammatory drugs at day 0. E) A treatment to a chronic wound (k3 =
0, R = 0.1) with k1 = 0 at t > day 3 representing the study by Mirza et al. [4] where anti-inflammatory drugs was delivered at day 3 post-injury.
[4] Mirza, Rita. Diabetes. 2013
Future Work
• Investigate the effect of sequential activation of macrophages in chronic wounds and in FBR to implants
• Explore how macrophages affect other cell types such as fibroblasts in wound healing and the FBR
• Improve the math model by incorporating other cell types and distinguishing the M2 subpopulations
Acknowledgements
The authors would like to acknowledge the collaboration with the Shanghai Jiao Tong University, the funding
from the Whitaker International Scholarship Program, and the assistance of the Kimmel Cancer Center
Consortium of Thomas Jefferson University