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Inflammatory Cell Responses to Vascular Regenerative Methacrylic Acid-Containing Materials
by
Kongyu David Zhang
A thesis submitted in conformity with the requirements for the degree of Masters of Applied Science
Institute of Biomaterials and Biomedical Engineering University of Toronto
© Copyright by Kongyu Zhang 2016
ii
Inflammatory Cell Responses to Vascular Regenerative
Methacrylic Acid-Containing Materials
Kongyu David Zhang
Master’s of Applied Science
Institute of Biomaterials and Biomedical Engineering
University of Toronto
2016
Abstract
Poly(methacrylic acid-co-methyl methacrylate) (MAA) beads improve vascularization when
applied to cutaneously-wounded diabetic mice. The aim of this thesis is to understand the
vascular regenerative properties of MAA at the cellular and molecular level. Subcutaneous
injection of MAA beads promoted the formation of a denser and perfusable network of blood
vessels at days 3 and 7 relative to poly(methyl methacrylate) (MM) control beads. MAA beads
modulated the host response; promoting more neutrophils at day 1 and more macrophages at day
7, relative to MM beads. A M2 macrophage polarization bias was observed in MAA-treated
animals but not in MM-treated animals. Additionally, complement was involved in the
mechanism of MAA; complement inhibition (at the C1 or C3 levels) diminished the M2
polarization bias at day 3, although no changes in vascularity were noted. These findings deepen
our understanding of MAA and benefit the development of MAA-based biomaterials for
applications in regenerative medicine.
iii
Acknowledgments
I owe my sincerest gratitude to the support and inspiration of many remarkable friends and
colleagues without which this work would not be possible. I am indebted to Prof. Michael Sefton
for giving me an opportunity to embark on this scientific journey over the last two years. I am
grateful for his unwavering support, continuous guidance, and astute criticism throughout the
entire course of my study. Thank you to my committee members, Prof. Warren Chan, Prof. Clint
Robbins, and Dr. Christoph Licht for their meticulous suggestions and for asking the important
questions to ensure that my research, presentation, and writing upholds the highest standard.
I am extremely fortunate and proud to be part of one of the greatest laboratories in the
world. Thank you to all the members of the Sefton Lab for supporting me, challenging me, and
helping to shape me into a better researcher and individual. Thank you to Sasha Lisovsky and
Dean Chamberlain for being excellent mentors, for pointing out the flaws in my experiments and
for making sure that I was asking the right questions. Thanks to Alexander Vlahos and Nicholas
Cober for the laughs, memories and helpful scientific discussions. Thank you also to Redouan
Mahou, Michael West, Gabrielle Lam, Ilana Talior-Volodarsky, Virginie Coindre, and Yarden
Gratch. A big thank you to Chuen Lo for his surgical wisdom and for all of our fascinating
discussions; this work would truly not be possible without him.
One of the best parts of working in such an interdisciplinary field is the opportunity to work
closely with individuals from many diverse fields. Thanks to the Chan, Wheeler, and Yip labs for
being such a fun crowd to work alongside. Thanks to Shrey Sindhwani for sharing his insight
and for the mentorship. Thank you to Wilson Poon for sharing his love of food and assimilating
me into the “foodie” culture. Some of our most productive scientific discussions occurred over
delicious (and sometimes, not so pleasant) meals.
Thank you to Dionne White for making flow cytometry enjoyable and to the PRP lab for sharing
all their histology-related expertise. Thank you to Prof. Penney Gilbert for giving me the
opportunity to teach and to Mohammad Saleh for teaching me how to become a better mentor.
Lastly, thank you to my parents and brother for encouraging me to pursue my passions,
regardless of what they are. This thesis is dedicated to my late grandfather, who instilled in me
the importance of higher education.
iv
Table of Contents
Acknowledgments.......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Figures ............................................................................................................................... vii
List of Appendices ....................................................................................................................... viii
List of Abbreviations ..................................................................................................................... ix
Chapter 1 Inflammatory cell responses to methacrylic acid beads ..................................................1
Introduction .................................................................................................................................1
1.1 The need for vascularization in regenerative medicine .......................................................1
1.2 Host response to biomaterial implantation ..........................................................................2
1.2.1 Neutrophils ...............................................................................................................3
1.2.2 Monocytes/Macrophages .........................................................................................3
1.2.3 Foreign body giant cells ...........................................................................................4
1.3 Role of macrophages in healing and vascularization ...........................................................5
1.4 Vascularizing biomaterials...................................................................................................7
1.5 Methacrylic acid-containing materials .................................................................................7
1.6 Objectives ............................................................................................................................9
Materials and Methods ..............................................................................................................10
2.1 MAA and MM bead preparation........................................................................................10
2.2 Subcutaneous injection animal model ...............................................................................10
2.3 Histology and immunohistochemistry ...............................................................................11
2.4 Tissue explant and digestion ..............................................................................................12
2.5 Analysis of cellular infiltrate in explanted tissues .............................................................12
2.6 CLARITY preparation and imaging ..................................................................................13
2.7 Statistical Analysis .............................................................................................................14
Results .......................................................................................................................................15
v
3.1 Subcutaneous injection model ...........................................................................................15
3.2 Effect of MAA beads on vascularization ...........................................................................15
3.3 Cellular response to MAA beads .......................................................................................17
3.3.1 Effect of MAA beads on the inflammatory cell infiltrate ......................................17
3.3.2 Effect of MAA beads on macrophage polarization ...............................................19
3.4 Interrogating biomaterial-cell interactions in intact tissues ...............................................21
3.4.1 Effect of MAA beads on CD206 expression in surrounding macrophages ...........22
Discussion .................................................................................................................................24
4.1 Effect of MAA beads on vessel formation ........................................................................24
4.2 Effect of MAA beads on the inflammatory cell infiltrate ..................................................24
4.3 Effect of MAA beads on macrophage polarization ...........................................................26
4.4 Insights into MAA-mediated macrophage polarization using CLARITY .........................27
Conclusion ................................................................................................................................30
Chapter 2 Role of complement activation in MAA-mediated macrophage polarization ..............31
Introduction ...............................................................................................................................31
1.1 Protein-biomaterial interactions in the host response ........................................................31
1.2 Mechanisms of macrophage recruitment and polarization ................................................33
1.2.1 Neutrophils .............................................................................................................34
1.2.2 Complement proteins .............................................................................................34
1.2.3 IGF signaling pathway ...........................................................................................34
1.3 Biomaterial strategies for mediating macrophage polarization .........................................35
1.4 Complement modulating effects of MAA .........................................................................35
1.5 Objectives ..........................................................................................................................37
Methods .....................................................................................................................................38
2.1 Preparation of poly(methacrylic acid-co-isodecyl acrylate) films .....................................38
2.2 Isolation, culture, and characterization of bone marrow-derived monocytes ....................38
vi
2.3 Macrophage stimulation by biomaterials in vitro ..............................................................39
2.4 CH50 type hemolysis assays ..............................................................................................39
2.5 Complement drug inhibition study ....................................................................................40
2.6 Tissue explant and digestion ..............................................................................................40
2.7 Analysis of cellular infiltrate in explanted tissues .............................................................41
2.8 Statistical Analyses ............................................................................................................41
Results .......................................................................................................................................42
3.1 Investigating the mechanism of MAA-mediated macrophage polarization ......................42
3.1.1 In vitro analysis of BMDM treated with MAA beads and films ...........................42
3.2 Inhibition of serum-derived complement and its effect on MAA ......................................42
3.2.1 Effect of complement inhibition on the vascular regenerative properties of
MAA ......................................................................................................................43
3.2.2 Effect of complement inhibition on MAA-mediated inflammatory cell
infiltration ..............................................................................................................45
3.2.3 Effect of complement inhibition on MAA-mediated M2 macrophage
polarization ............................................................................................................46
Discussion .................................................................................................................................50
4.1 Role of complement inhibition in MAA-mediated vascularization ...................................50
4.2 Role of complement inhibition in MAA-mediated alternative host response ...................51
4.3 Role of complement inhibition in MAA-mediated M2 macrophage polarization .............52
4.4 Insight into the mechanism of vascular regenerative MAA beads ....................................54
Conclusion ................................................................................................................................55
References ......................................................................................................................................57
Appendices .....................................................................................................................................64
vii
List of Figures
Fig. 1. The host response to biomaterial implantation.
Fig. 2. Role of macrophages in the host response and vascularization.
Fig. 3. Subcutaneous injection mouse model.
Fig. 4. MAA beads induced formation of perfusable vessels when injected subcutaneously.
Fig. 5. No differences in the density of F4/80+ cells between MAA- or MM- treated animals.
Fig. 6. Treatment with MAA beads altered the inflammatory cell landscape.
Fig. 7. Treatment with MAA beads biased macrophages towards a M2 polarization state.
Fig. 8. More CD206+ macrophages are found in the vicinity of MAA beads relative to MM
beads.
Fig. 9. Drug-induced inhibition of complement activation.
Fig. 10. Administration of pentamidine and ATA inhibited complement activation.
Fig. 11. Inhibition of complement activation did not affect the vascular potency of MAA.
Fig. 12. Complement inhibition eliminated MAA’s neutrophil recruitment effect.
Fig. 13. Complement inhibition altered the MAA-mediated effects on M2 macrophage
polarization.
Fig. 14. Effect of MAA beads on macrophage polarization, vascularization and the role of
complement activation.
viii
List of Appendices
S1. Gating strategy for macrophages (day 3 shown; MAA beads).
S2. Markers used for immunohistochemistry and flow cytometry analyses and definitions.
S3. Explant mass, cell number, and normalized cell number for flow cytometry analyses.
S4. Leukocytes, endothelial and dendritic cell populations in explanted tissues.
S5. Expression of CD206, CD86, and MHCII in bone marrow-derived macrophages polarized by
IFNγ and IL-4.
S6. Macrophage polarization - single positive cells.
S7. Formation of giant-like cells in vitro.
S8. Gating strategy for validating dextran uptake in CD206+ macrophages.
S9. Bone marrow harvest, macrophage culture and treatment with MAA beads or films.
S10. Gating strategy for macrophages following complement inhibition (day 7 shown; MAA
beads).
S11. MAA beads increased CD206, but not MHCII expression in the presence of blood.
S12. MAA films stimulated M2 marker Arg1 in M0 and M(IFNγ) cells.
S13. Administration of 4 mg/kg pentamidine or 2.5- 10 mg/kg ATA did not inhibit complement
activation over time.
S14. Explant mass, cell number and normalized cell number in complement-inhibited animals.
S15. Leukocytes and macrophage populations in complement-inhibited animals.
S16. Published manuscript: Lisovsky A, Zhang, DKY, Sefton MV, Biomaterials 2016.
S17. Curriculum vitae.
ix
List of Abbreviations
ATA: aurin tricarboxylic acid
ATP: adenosine triphosphate
Cx,y: (number) complement component, subunit (number)
CDxx: cluster of differentiation (number)
CLARITY: Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining /
in situ-hybridization-compatible Tissue hYdrogel
CSF: colony stimulating factor
DAMPS: danger associated molecular patterns
DNA: deoxyribonucleic acid
ECM: extracellular matrix
FBGC: foreign body giant cells
FBS: fetal bovine serum
GSL/BSL: Griffonia (Bandeiraea) Simplicifolia lectin
HUVEC: human umbilical vein endothelial cell
IFNγ: interferon gamma
IL-x: interleukin (number)
LAL: limulus amebocyte lysate
Ly6G: lymphocyte antigen 6 complex, class G
MAA: methacrylic acid
MAA beads: poly (methacrylic acid-co-methyl methacrylate) beads
MCP: monocyte chemoattractant protein
MHC: major histocompatibility complex
MM: methyl methacrylate
MM beads: poly (methyl methacrylate) beads
NOS: nitric oxide synthase
PBS: phosphate buffered saline
PDGF: platelet-derived growth factor
PEG: polyethylene glycol
TGF: transforming growth factor
TNF: tumor necrosis factor
VEGF: vascular endothelial growth factor
1
Chapter 1 Inflammatory cell responses to methacrylic acid beads
Introduction
Biomaterials are substances designed to interface with biological systems[1]. The past half-
century represents a “biomaterials revolution”; advancements in the development of biomaterials
for drug delivery (e.g., microcapsules, tablets), surgery (e.g., sutures, adhesives), and implants
(e.g., prostheses, vascular grafts) are promised to innovate modern medicine[2]. While
significant progress has been made in the development of “interesting” biomaterials[3], there
remains an incomplete understanding of the interactions between an implanted material and
biological tissues. The conventional interpretation of these interactions begins at the protein-
adsorption level. An adsorbed layer of proteins dictates changes in cell behavior and the
activation of blood-derived pathways. Together, these interactions translate into inflammation,
vascularization, and ultimately, tissue remodeling [4–6]. Here, we explore the biological
interactions between methacrylic acid (MAA)-based biomaterials, the host response and
vascularization. These materials have a vascular regenerative effect in vivo [7], through an
unclear mechanism. The present chapter evaluates the host response to MAA-containing
polymeric beads and aims to define a role for macrophage polarization in this context. Much of
this chapter has been published (See Appendix); the introduction, discussion, and parts of the
results have been expanded for the purpose of this thesis. Chapter 2 pursues mechanistic
questions, with a focus on complement activation, and aims to connect the events that occur
immediately following biomaterial implantation to the changes in the host response (i.e.,
inflammatory cell infiltration) and vascularization. Understanding of the mechanisms behind
MAA-mediated vascularization may afford the ability to control and dictate the biological
response to similar materials.
1.1 The need for vascularization in regenerative medicine
A perfusable network of blood vessels is vital for regenerative medicine[7,8]. In cell therapy, the
transplantation of therapeutic cells requires a vascularized network to ensure that nutrients and
oxygen are aptly delivered[8]. In the context of tissue regeneration, the development of a
vascular network stimulates endogenous repair by delivering growth factors to the site of
injury[9]. To meet this need for vascularization, a number of approaches have been devised[8].
2
However, most strategies employ the synergistic addition of vascular support cells (e.g.,
mesenchymal stromal cells)[10] or growth factors (e.g., VEGF)[11,12], leading to complicated
and costly treatments that are difficult to translate to the clinic[13]. This poses a unique
opportunity for the development of alternative, scalable and more cost-effective strategies (i.e.,
biomaterials) to address this need. Biomaterials that promote vascularization without the co-
delivery of cells or proteins would be highly advantageous, as they would be cost-effective and
easy to manufacture. Studies in wounded diabetic mice revealed that materials containing
methacrylic acid (MAA) have vascular regenerative properties[14–17]. The aim of this thesis is
to investigate the mechanism behind this beneficial effect at the cellular and molecular levels.
1.2 Host response to biomaterial implantation
One prominent issue with the use of biomaterials for improving vascularization is the host
response or foreign body response[6]. In the process of biomaterial implantation, cells and
tissues are inevitably damaged, setting the stage for the multitude of interactions collectively
known as the host response[18]. The host response is a generic biological response that begins
with inflammation and ends with fibrosis or tissue reconstitution and healing [5]. Following
biomaterial implantation, tissue-resident cells (e.g., tissue-resident macrophages) and blood-
derived proteins (e.g., complement) detect the presence of the foreign material indirectly via
damage-associated molecular patterns (DAMPS), such as cytoplasmic proteins (e.g., ATP, DNA,
uric acid, etc.) released from dying cells, or directly, via the non-specific adsorption of proteins
to the biomaterial itself[6,19,20]. The adsorbed proteins are dynamically changing and are
thought to dictate the outcome of the host response[5]. Concomitantly with protein adsorption,
damage to blood vessels initiates thrombosis and the formation of a fibrin clot; a process
involving platelets, the complement system, the fibrinolytic system, and others (reviewed in
[21]). Together, these processes facilitate the formation of a provisional matrix; a rich, dynamic
ecosystem of chemokines, cytokines, and growth factors that modulates cell activation and
proliferation in the inflammatory and healing phases of the host response[6]. Following
provisional matrix formation, inflammatory cues (e.g., IL-1β, TNF-α, and others) derived from
various sources including de-granulated mast cells, are released from the matrix into the blood
stream to signal the recruitment of innate immune cells (e.g., neutrophils, monocytes,
lymphocytes) to the site of the foreign material or the site of inflammation[5].
3
1.2.1 Neutrophils
The cell type dominating the host response is time-dependent[22]. Neutrophils are the hallmarks
of the inflammatory response and traditionally the first responders to a site of injury[23] (Fig. 1).
Neutrophils are short-lived cells (24-48h) whose primary function is to remove and contain the
spread of foreign particles (e.g., pathogens or debris, reviewed in [22]). Being myeloid-derived
cells (i.e., cells that express CD11b; cluster of differentiation 11b, an integrin associated with
leukocyte adhesion), they also express Ly6G (lymphocyte antigen 6 complex, class G), a GPI-
linked differentiation antigen at varying levels corresponding to their maturity[24]. Once the task
of “quarantining” foreign particles from the rest of the body is completed, neutrophils become
apoptotic, inhibiting further neutrophil recruitment while promoting monocyte recruitment and
their subsequent differentiation to macrophages (reviewed in [25–28]). This feedback loop
enables apoptotic neutrophils to be phagocytosed by growing numbers of macrophages that have
infiltrated the site of inflammation.
1.2.2 Monocytes/Macrophages
Monocytes responding to gradients of granulocyte (e.g., neutrophil, eosinophil, basophil) –
derived chemokines (e.g., MCP-1, IL-1, etc.) hone in to the site of the foreign material[22]. Once
these myeloid-derived innate immune cells leave the blood vessel, their differentiation to
macrophages is triggered, as noted by an upregulation in the expression of F4/80 (an adhesion G-
coupled protein receptor associated with peripheral T cell tolerance)[29]. A positive feedback
loop propagates further secretion of chemokines, such as granulocyte colony stimulating factor
(G-CSF), promoting more macrophage infiltration. At the site of the biomaterial, macrophages
serve multi-faceted roles and link the inflammatory and healing phases of the host
response[30,31]. The literature suggests that there are two distinct subsets of macrophages
(termed M1 and M2); however, this represents an oversimplification of a complex spectrum of
macrophage polarization states[32]. Macrophages are highly multi-functional and the M1/M2
classification is an in vitro artifact that represents the ends of a spectrum of phenotype and
function[33].
Following tissue infiltration and activation, macrophages promote inflammation by secreting
cytokines such as IL-1β and TNF-α – a property characteristic of the M1 polarization state (Fig.
2A). Later in the host response, macrophages secrete IL-10 and TGF-β, paving the road for the
4
resolution of inflammation – a property characteristic of the M2 polarization state (Fig. 2A). It is
accepted that macrophages shift from the M1 to the M2 phenotype 48-72 h post-injury,
coordinating the transition from the inflammatory phase to the healing phase of the host
response[34,35]. Consistent with this idea, studies involving fluorescently labeled macrophages
revealed that a part of the population of M2 macrophages that arises later in the host response is
derived directly from the original M1 macrophage population at the site of the foreign
material[36,37].
1.2.3 Foreign body giant cells
At later time points (several days), adherent macrophages on the surface of a biomaterial fuse to
form foreign body giant cells (FBGCs)[5]. The shift in macrophage polarization from M1 to M2
is expected; studies from J. Anderson et al indicated that FBGC formation requires IL-4 and IL-
13, agonists of the M2 phenotype[38]. However, the genomic and proteomic expression profile
of FBGC is distinct from M2 macrophages[38], highlighting the FBGC as a distinct phenotype.
FBGC formation is in a sense a cellular stress response to large foreign bodies and is a
conventional response to biomaterial implantation. Although macrophages are capable of
phagocytosing small particles (<5 μm), once they encounter a larger particle (>10 μm), they fuse
to increase their combined surface area and corresponding phagocytic potential[38]. If the
newly-formed FBGCs are unable to phagocytose the foreign material, they remain at the
biomaterial-host environment and attempt to degrade the foreign material instead via the
secretion of matrix metalloproteinases (MMPs), protons, and reactive oxygen species (ROS)[6].
Thus, FBGCs form an isolated degradative environment that may lead to 1) biomaterial
resorption and the resolution of the host response[6], if the biomaterial is degradable or 2)
persistent inflammation and evidence of chronic inflammation[5,39], if the biomaterial is not
degradable.
Successful tissue regeneration is associated with a milieu of anti-inflammatory mediators,
downregulation of inflammatory mediators and the apoptosis of immune cells; which
collectively mediates the resolution of inflammation[28]. FBGC that have failed to degrade the
foreign material secrete pro-fibrotic factors, such as TGF-β, recruiting and activating fibroblasts
to deposit collagen and remodel the extracellular matrix[5]; forming the underpinnings of the
5
fibrotic capsule[38]. Thus, biomaterial-adherent macrophages dictate the fibrotic response and
the formation of a fibrotic capsule, the conventional endpoint of biomaterial implantation.
Fig. 1. The host response to biomaterial implantation. Following implantation of a
biomaterial, a spike in neutrophils is observed at the site of the foreign material, followed by
macrophage infiltration and the beginnings of neovascularization (the formation of new vessels),
then the formation of foreign body giant cells, the recruitment of fibroblasts, and ultimately, the
formation of a fibrotic capsule. The y-axis (intensity) may be interpreted as the number of cells.
The scale of the x-axis (time) varies depending on the biomaterial; for most biomaterials, the
initial wave of neutrophils is resolved in 48h and fibrosis occurs several weeks after
implantation. Adapted from [18].
1.3 Role of macrophages in healing and vascularization
As an alternative to fibrosis, the host response can also prepare the ground for tissue regeneration
and vascularization[9]. Macrophages lie at the crossroads of inflammation, tissue regeneration
and vascularization[30]. The specific contributions of classically-activated (M1) and
alternatively-activated (M2) macrophages in the vascularization process are ill-defined; some
studies showed that lower ratios of M1/M2 macrophages improves vascularization[39], while
others have shown that increased M1/M2 ratios leads to more vascularization[40]. One
hypothesis claims that vascularization begins with classically-activated (M1) macrophages, a
potent source of vascular endothelial growth factor (VEGF), which initiates vessel sprouting in
responding endothelial cells[41,42] (Fig. 2B). Endothelial cells migrate and sprout outwards,
while secreting integrins and creating new extra-cellular matrix (ECM), forming a leaky and
immature vasculature.
6
Fig. 2. Role of macrophages in the host response and vascularization. (A) Blood-derived
monocytes infiltrate the site of inflammation, triggering their differentiation to macrophages.
Over time, macrophages shift from an inflammatory M1 to an anti-inflammatory M2 phenotype
directly at the site of inflammation. (B) Proposed roles of M1 and M2 macrophages in
vascularization. M1 macrophages secrete endothelial growth factors to initiate vessel sprouting
(via VEGF) while M2 macrophages promote vessel maturation by recruiting pericytes via
PDGF.
Eventually, as macrophages transition to the M2 polarization state, these alternatively-activated
(M2) macrophages aid in the degradation of the basal lamina by secreting metalloproteinases
(MMPs) to break down the surrounding extracellular matrix. These cells serve as chaperones for
endothelial cells by guiding tip cell anastomosis – the process of joining of the ends of two
newly formed vessels[11,43]. Additionally, M2 macrophages promote the maturation of vessels
through the recruitment of pericytes via the secretion of platelet-derived growth factor
(PDGF)[11]. Simply put, M2 macrophages mature the leaky vasculature formed by M1
macrophages, supporting the formation of a perfusable and mature vascular network[44].
7
Hematopoietic and mesenchymal-derived progenitor cells are also sources of VEGF and PDGF,
integrating themselves into vessels, among other tissue structures[45,46]. Fibroblasts also play a
role in laying down the foundation for vessels by synthesizing the extracellular matrix. In
summary, vascularization is a complex process that is intimately linked to the host response and
requires the synergistic contributions of both M1 and M2 macrophages.
1.4 Vascularizing biomaterials
Biomaterials have been used to deliver cells (e.g., vascular support cells; adMSC)[10] or growth
factors (e.g., VEGF). However, cell delivery strategies become increasingly complex with a
number of immunological barriers (e.g., inflammation, antigen-directed cytotoxic cell responses)
to overcome[47]. While the delivery of growth factors is simpler, it remains challenging to
temporally control growth factors to 1) initiate vessel formation and 2) mature the newly-formed
vessels. Attempts have been made to modulate the host response to benefit vascularization.
Madden et al showed that increasing porosity in acellular poly(2-hydroxyethl methacrylate)
[poly(HEMA)] scaffolds promoted cellular infiltration and facilitated vascularization[42]. They
attributed this beneficial effect to macrophage polarization; macrophages recruited to the
poly(HEMA) scaffold were polarized towards a “healing” phenotype, characterized by an
increase in the expression of CD206. Stupp et al used a bio-inspired approach to develop peptide
nanostructures that mimicked VEGF activity. The VEGF-like peptides bound to VEGF receptors
and initiated vessel sprouting in endothelial cells[48]. Biomaterials that have the ability to alter
the cellular landscape to promote regeneration have the potential to compete and replace
cell/protein-based strategies of vascularization.
1.5 Methacrylic acid-containing materials
Methacrylic acid (MAA)-based biomaterials were shown to have a vascular regenerative effect
in the absence of exogenous cells or growth factors[7]. These biomaterials promoted
vascularization[14,16,49], myocutaneous graft survival[14], and diabetic wound healing[16]. In
vitro studies[50,51] involving human endothelial cells (i.e., HUVEC) did not alter the expression
of classical angiogenic genes (i.e., VEGF)[51]. However, gene expression analysis revealed that
MAA modulated pleiotropic genes (i.e., Shh), pro-inflammatory genes (i.e., IL-1β, TNF-α), in
bone marrow-derived macrophages and macrophage-like cells (dTHP-1), as well as in diabetic
wounds and in an air pouch model[17,49,50]. More recently, a phosphoproteomics study with
8
dTHP-1 cells treated with MAA-based material highlighted a number of phosphorylated proteins
involved in macrophage polarization[52] among several hundred proteins that were differentially
phosphorylated between a MAA-based and a control (methyl methacrylate-based) material[53].
In the studies conducted to date, no change was observed in the number of infiltrating
macrophages between MAA-treated animals or the control MM-treated animals [17]. These data
led to the hypothesis that MAA elicited its vascular regenerative effect by modulating
inflammatory cell responses, specifically macrophage polarization.
Here, a subcutaneous injection model was devised to investigate the effects of MAA on the host
response and macrophage polarization. To this end, poly(methacrylic acid-co-methyl
methacrylate) (MAA) beads and control poly(methyl methacrylate) (MM) beads were injected
subcutaneously in male CD1 mice. The bead explants were processed for immunohistochemistry
and flow cytometry for the number of cells and the polarization state of macrophages. MAA
beads increased the density of neutrophils at day 1, macrophages at day 7 and biased
macrophages towards the MHCII-CD206+ state, representative of the “M2” phenotype.
9
1.6 Objectives
This thesis explores the interactions between methacrylic acid (MAA)-containing beads and the
host response. Chapter 1 investigates the effect of MAA beads on the inflammatory cell
response. As macrophages are known to be the orchestrators of vascularization, they were a
focus of the investigation. Macrophage polarization was studied using 1) flow cytometry to
evaluate global changes in macrophage phenotype in response to MAA bead implantation, and 2)
a 3D tissue imaging approach to interrogate local changes in macrophage polarization in the
immediate vicinity of MAA beads. Chapter 2 (Aim 2) investigates the role of complement
activation in MAA-mediated macrophage polarization and vascularization.
Aim 1A: Characterize the inflammatory cell infiltrate in animals injected subcutaneously with
MAA beads.
Hypothesis: Treatment with MAA beads alters the inflammatory cell landscape and alters
macrophage polarization relative to control MM beads.
Aim 1B: Interrogating MAA bead-cell interactions in intact tissues using CLARITY, a tissue
preparation protocol for 3D imaging of intact tissues.
Hypothesis: MAA beads polarize macrophages in its immediate vicinity (< 200 μm distance
from a cluster of MAA beads).
10
Materials and Methods
2.1 MAA and MM bead preparation
Poly(methacrylic acid-co-methyl methacrylate) (MAA-co-MMA or MAA) beads were composed
of 45 mol% methacrylic acid (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada), 1 mol%
ethylene glycol dimethacrylate (Sigma-Aldrich Canada Ltd.) and 54 mol% methyl methacrylate
(Sigma-Aldrich Canada Ltd.). MAA beads were synthesized by suspension polymerization as
previously described and were sieved to obtain beads in the diameter range of 150-250 μm.
Methacrylic acid content of the synthesized beads was confirmed by titration. Control
poly(methyl methacrylate) (MM) beads (same diameter) were obtained from Polysciences
(Warrington, PA). Beads were washed in either 95% ethanol (MAA beads) or 1 N HCl (MM
beads) repeatedly and then rinsed five times in LAL reagent water (MJS Biolynx Inc.,
Brockville, ON, Canada) prior to use in vivo. Analysis with a limulus amebocyte lysate (LAL)
pyrochrome endotoxin test kit (Cape Cod Inc., Falmouth, MA) indicated that beads contained
<0.25 EU/100 mg. MAA beads had a rough, porous surface, were negatively charged and non-
degradable; MM beads were smooth and also not degradable.
For subcutaneous injections, a 1 mL syringe with an 18-gauge needle was loaded with either 5
mg MAA beads or 15 mg MM beads (or no beads, vehicle control) suspended in 250 μL of 50%
w/v polyethylene glycol (PEG, avg. mol. wt. 1450, sterile-filtered; Sigma-Aldrich Canada Ltd.)
in PBS. The 1:3 weight ratio (5 mg MAA: 15 mg MM) was used to account for MAA beads
swelling upon hydration at physiological pH to approximately equate implanted volumes. The
vehicle control was used only for flow cytometry analysis because the vehicle control implant
area could not be defined reproducibly for vessel and cell density analyses.
2.2 Subcutaneous injection animal model
Mice were anesthetized with 0.5% w/v isofluorane prior to surgery and an analgesic
(Ketoprofen, 5 mg/kg) was administered intraoperatively. The dorsal area of a mouse was shaved
and the remaining hair was removed by hair removal cream (Veet). The skin was sterilized with
70% ethanol and Betadine. An 18-gauge needle was used to inject MAA, control MM beads or
vehicle (PEG). Two injections on either side of the dorsum were performed for each mouse. A
small subcutaneous pocket was made with the needle on the side of the dorsum by moving the
11
syringe from side to side, while deliberately attempting to nick small blood vessels to promote
injury prior to injection (Fig. 3). Following surgery, mice were housed individually, fed chow
and water ad libitum, and monitored for any signs of discomfort. At 1 to 7 days post-injection,
the mice were sacrificed using CO2, followed by cervical dislocation. The implants were
removed surgically and processed for histology, imaging or flow cytometry. All animal work
was done with the approval of the University of Toronto Animal Care Committee. Animals were
housed under sterile conditions in the University of Toronto’s Department of Comparative
Medicine (AUP #20010994 and #20013994).
Fig. 3. Subcutaneous injection mouse model. CD1 mice were injected subcutaneously with
MAA or MM beads. At days 1, 3, and 7 post-implantation, the beads and surrounding tissues
were excised and processed for histological, imaging, and flow cytometry analyses. The
molecular analyses panel is included as it is a future possibility of this model; molecular analysis
was not conducted in the present study. In addition, this model may be used to explore biological
responses to other biomaterials.
2.3 Histology and immunohistochemistry
Immediately upon euthanizing mice, the bead implant and several mm of surrounding tissue was
excised from the right side of the dorsum and fixed in formalin. Tissue samples were embedded
in deep paraffin blocks, cut into sections, processed and stained with hematoxylin and eosin
(H&E), Masson's trichrome, CD31 and F4/80 (Appendix, S2). Histology slides were scanned
(20x) using an Aperio ScanScope XT (Leica Microsystems, Concord, ON, Canada) by the
Advanced Optical Microscopy Facility (AOMF, Toronto, ON, Canada).
12
The scanned slides were analyzed using Aperio ImageScope (Version 11) at 3 and 7 days post-
implantation. Vessel and cell quantitation was performed by first defining a region of interest
(ROI). For vessel counts, the ROI was defined by measuring a distance of 500 μm around a
hotspot (a clump of beads with CD31+ vessels in its ROI; some clumps of beads had no vessels
in its ROI). CD31+ vessel-like structures (criterion being the presence of a lumen) were counted
in the tissue within this defined region. The vessel density was calculated by dividing the total
number of vessels by area of the ROI. For F4/80+ cell counts, a distance of 200 μm around each
bead cluster was used to define the ROI.
2.4 Tissue explant and digestion
Subcutaneous tissue containing the injected beads was separated from the skin and muscle
layers. For PEG samples, subcutaneous tissue was explanted in the same manner using the
injection needle wound site as a guide. Tissues were weighed and then digested following a
previously described digestion protocol [54]. Briefly, samples were finely minced in 500 µL of 1
X HBSS containing 450 U/mL collagenase I (Sigma-Aldrich Canada Ltd.), 125 U/mL
collagenase XI (Sigma-Aldrich Canada Ltd.), 60 U/mL DNase I (Sigma-Aldrich Canada Ltd.),
60 U/mL hyaluronidase (Sigma-Aldrich Canada Ltd.) and 20 mM HEPES (Sigma-Aldrich
Canada Ltd.). The samples were homogenized using a gentleMACS Octo Dissociator (Miltenyi
Biotec Inc., San Diego, CA). The tissues were further digested for 60 min at 37 °C and 250 rpm.
The cell suspension was filtered using a 40 μm cell strainer (Fisher Scientific, Ottawa, ON,
Canada) to remove beads and debris. The remaining cells were washed in PBS supplemented
with 0.5% BSA and 2 mM EDTA, pelleted and stained with live/dead stain, CD11b, CD206,
CD11c, CD31, CD45, CD86, F4/80, Ly6G, and MHCII (Appendix, S2). All antibodies were
diluted according to the manufacturers’ recommendations and titrated in-house to optimize
staining.
2.5 Analysis of cellular infiltrate in explanted tissues
The gating strategy (Appendix, S1) was as follows: after isolating live single cells, CD45
distinguished leukocytes from non-leukocytes. Neutrophils were identified as Ly6G+ and
dendritic cells as CD11c+Ly6G-. Macrophages were first identified as CD11c-Ly6G-
F4/80+CD11b+ and then further characterized as MHCII+CD206- (“M1”) and MHCII-CD206+
(“M2”). Endothelial cells were identified as CD31+CD45-. Cells were gated according to
13
positive staining for each antibody using fluorescence minus one (FMO) controls. Cell
populations were expressed as either a percentage or as a normalized value (estimated total
number of cells divided by the weight of the explanted tissue). The number of cells was
estimated from the flow cytometry results with 123count eBeads (eBioscience) used to determine
cell recovery (~50%).
2.6 CLARITY preparation and imaging
Seven days following subcutaneous injection of MAA or MM beads, Cy5-conjugated dextran
(70kDa; 100 μg in 150 μL PBS; Chan lab) was injected via tail vein. Dextran is the ligand for the
CD206 scavenger receptor[55]. After 30 min of circulation, Alexa 555-conjugated lectin (GSL-
1: Griffonia (Bandeiraea) Simplicifolia; 100 μg in 150 μL PBS; Vector Laboratories, Burlington,
ON, Canada) was injected via tail vein prior to sacrifice and whole body perfusion with PBS-
heparin[56]. Fluorophore conjugation was performed in-house using Alexa 555 or Cy5 modified
with a NHS-ester chemistry[57]. GSL-1 is a lectin which binds to the galactosyl residues of
mouse endothelial cells, enabling labeling and visualization of the mouse vasculature[58]. Earlier
experiments were performed without the initial injection of Cy5-conjugated dextran. The
implants with the surrounding subcutaneous tissue were removed surgically and processed using
a modified CLARITY protocol developed by Sindwani, S. et al [56,59]. Briefly, explants were
fixed in a solution containing 2% acrylamide (Sigma-Aldrich Canada Ltd.), 4%
paraformaldehyde (Sigma-Aldrich Canada Ltd.) and 0.25% (w/v) VA-044 thermal initiator
(Sigma-Aldrich Canada Ltd). After one week of incubation, the acrylamide was polymerized at
37 °C for 1-3 h. Polyacrylamide-embedded explants were cleared for 14 days at 50°C in clearing
solution (8% SDS in borate buffer, pH 8.5; eBioscience, San Diego, CA), which was changed
every 2nd day. Post-clearing, the explants were counterstained with SYTOX green nucleic acid
stain (100 pmol/mg; Life Technologies, Burlington, ON, Canada) or DAPI nucleic acid stain
(200 pmol/mg; Life Technologies, Burlington, ON, Canada) for 48 hours. Refractive index
matching was performed by infusing explants with 70% 2,2’-thiodiethanol[56] in borate (Sigma-
Aldrich Canada Ltd.) for confocal microscopy. Explants were imaged using a Nikon A1 confocal
microscope (Nikon, Melville, NY) at the Center for Microfluidics Systems (University of
Toronto).
14
2.7 Statistical Analysis
Statistical analysis was performed using GraphPad PRISM 6.0. Data is represented as mean ±
standard error of mean (SEM). A two-way ANOVA was used to compare treatment groups over
the 3 time points. Tukey’s post hoc test was used to determine significance of multiple
comparisons. A p-value of less than 0.05 was considered significant.
15
Results
3.1 Subcutaneous injection model
Previously, the vascular potency of MAA was explored in cutaneous wounded diabetic db/db
mice, precluding the analysis of the inflammatory cell infiltrate, as recruited cells became
entrapped in scabs. The less-invasive subcutaneous injection model obviated this issue and
enabled the direct interrogation of cells that were associated with MAA beads (See Fig. 3).
Additionally, the subcutaneous model simplified the host response, as there was less of a
physiological need for wound healing and the complexities of diabetic wound healing were
removed. MAA beads were subcutaneously injected in the dorsal flank of male CD1 mice at two
sites; one implant was harvested for flow cytometry analysis while the other was processed for
histological analysis.
3.2 Effect of MAA beads on vascularization
MAA beads enhanced vascularization in the tissue directly surrounding the beads (< 500 μm
from a cluster of MAA beads) following subcutaneous injection. CD31+ vessel formation was
increased at days 3 and 7, relative to MM beads (Fig. 4A, B), validating the vascular
regenerative effect of MAA in the subcutaneous injection model. To confirm that the MAA-
induced vessels were perfusable, animals were injected with Alexa 647-conjugated mouse lectin
(GSL1), to visualize blood vessels in the immediate vicinity of the beads (Fig. 4C). A modified
CLARITY protocol was used to increase the depth of imaging. This was the first application of
CLARITY to visualize biomaterial-cell interactions, to our knowledge. MAA-treated animals
showed high levels of GSL1 staining around MAA beads, consistent with the greater CD31+
vascularity observed in the histological analysis (Fig. 4C). On the other hand, MM-treated
animals showed minimal or no lectin staining surround MM beads. Instead, lectin staining was
primarily concentrated to the panniculus carnosus of the skin layer (Fig. 4C).
16
Fig. 4. MAA beads induced formation of perfusable vessels when injected subcutaneously.
(A) Histology sections of animals treated with MAA or MM beads at day 7 stained with CD31
(left) and Masson’s trichrome (right). Arrows indicate examples of vessels. (B) Tissues treated
with MAA beads in mice had a significantly higher vessel density at day 7. (C) Confocal
microscopy image of CLARITY-processed tissues treated with MAA and MM beads from non-
transgenic CD1 mice stained with Alexa 647-GSL1, a lectin specific for the mouse endothelium,
and Sytox Green. Perfused vessels weaved around MAA beads but not MM beads. Most of the
vessels in MM-treated mice were found in the skin further away from the beads. Scale bars = 200
μm. n = 3-4.
In CLARITY processed tissues, a thick layer of cells was found surrounding MM, but not MAA
beads, suggesting a differential cellular response to the MAA beads. This dense layer of cells
was also observed in trichrome-stained sections (Fig. 4A), indicating the presence of a
conventional host response to biomaterial implantation. Together, these data suggested that the
MAA beads induced vascularization when injected subcutaneously and that the MAA-induced
vessels were perfusable.
17
3.3 Cellular response to MAA beads
As macrophages play a vital role in vascularization[39], the effect of MAA on macrophages was
investigated in histological sections using the pan macrophage marker F4/80. A dense ring of
F4/80+ cells was found surrounding MM beads; but rarely surrounding MAA beads (Fig. 5A),
similar to that seen in trichrome-stained sections and CLARITY-processed tissues (Fig. 4A, C).
The thick ring of cells resembled foreign body giant cells. No differences were observed in
F4/80+ cell density between MAA or MM beads at day 3 or day 7 (Fig. 5B).
Fig. 5. No difference in the
density of F4/80+ cells
between MAA- or MM-
treated animals. (A) Tissue
sections from MAA- and
MM-treated mice stained
with pan macrophage F4/80
marker at day 3. A dense ring
of F4/80+ cells
(macrophages) surrounded
control MM beads. Arrows
show examples of cells
positive for the marker of
interest. (B) Density of
F4/80+ cells in tissues
following treatment with
MAA and MM beads. Scale
bars = 200 μm. n = 3-4.
3.3.1 Effect of MAA beads on the inflammatory cell infiltrate
Flow cytometry was used to follow up on the histological analysis. An extra time point (day 1)
was added to investigate the host response (i.e., neutrophils) immediately following
subcutaneous injection of MAA and MM beads. Also, a PEG vehicle treatment was added. No
statistical difference was noted between the mass of the explanted tissues, or the estimated total
cell number, or the normalized cell numbers among the three treatment groups (MAA beads,
MM beads, and PEG vehicle) at the studied time points (Appendix, S3). The gating strategy
employed is illustrated in Appendix, S1. Higher densities of CD45+ cells were found in the
harvested MAA implants relative to the PEG vehicle control at day 1, with a corresponding
18
higher density of CD45- non-leukocytes in the PEG vehicle implant (Fig. 6A, B; Appendix, S4).
Interestingly, a progressive increase in CD45- cells were noted in tissues treated with MM
relative to MAA beads at day 7 (Fig. 6B).
Fig. 6. Treatment with MAA beads altered the inflammatory cell landscape. (A-D) Number
of CD45+ leukocytes (A), CD45- non-leukocytes (B), Ly6G+CD11b+CD45+ neutrophils (C)
and F4/80+CD11c-Ly6G-CD11b+CD45+ macrophages (D). MAA beads significantly increased
the number of CD45+ cells (A) and neutrophils (C) at day 1 and macrophages at day 7 (D), while
decreasing the number of CD45- cells at day 7 (B). (E) F4/80 and CD11b expression in CD45+
cells at day 1 and day 7; note the F4/80 mean fluorescent intensity increased over time. The
F4/80 and CD11b gate (black box) was set based on fluorescence minus one (FMO) negative
controls. n = 3-4.
In the biomaterial treatment groups (MAA and MM), neutrophils were most prevalent at day 1
post-injection and their numbers dwindled by days 3 and 7. Treatment with MAA beads
increased the number of neutrophils relative to MM and PEG controls at day 1 (Fig. 6C). More
macrophages were found in explants harvested from MAA-treated mice at day 7, relative to
MM- and PEG- treated animals. Indeed, the estimated number of macrophages decreased from
day 3 to day 7 in MM- and PEG-treated animals; while the number remained unchanged in
MAA-treated animals (Fig. 6D). Additionally, the intensity of F4/80 expression varied greatly
from day 1 to days 3 and 7, suggesting that macrophages “matured” in the subcutaneous
injection site. Endothelial and dendritic cells were also quantified by flow cytometry, although
19
no significant differences were noted for the density of endothelial cells among the three
treatment groups (Appendix, S4). The frequency (as a % of Ly6G-CD45+ cells) of dendritic
cells increased in the material treatment groups, relative to PEG vehicle at day 7 (Appendix, S4).
Together, the data suggests that MAA altered the inflammatory cell response compared to MM
and PEG controls, leading to an increase in the recruitment of neutrophils at day 1 and the
number of macrophages at day 7.
3.3.2 Effect of MAA beads on macrophage polarization
Next, the flow cytometry protocol was used to distinguish macrophage polarization states.
MHCII and CD86 were used as markers for “M1”, classically-activated macrophages, while
CD206 was used as a marker for “M2”, alternatively-activated macrophages. These markers
were validated with bone marrow-derived macrophages stimulated with IFNγ and IL-4 for “M1”
and “M2” macrophages, respectively (Appendix, S5). In the subcutaneous injection model, the
expression of CD86 did not change significantly between treatment groups at any of the studied
time points; it was dropped from further analysis. There were some significant differences in the
expression of MHCII and CD206, with a general trend involving more CD206+ expression in
MAA-treated animals and more MHCII+ expression in MM-treated animals (Appendix, S6).
20
Fig. 7. Treatment with MAA beads biased macrophages towards a M2 polarization state.
(A, B) Representative dot plot of F4/80+ cells (macrophages) at day 3 in mice treated with MAA
(A) and MM beads (B). (C, D) The number and frequency of the individual single positive,
double positive, and double negative MHCII or CD206 macrophage populations in mice treated
with MAA beads, MM beads or PEG vehicle control. (C) Normalized number and frequency of
MHCII-CD206+ (“M2”) macrophages. (D) Normalized number and frequency of
MHCII+CD206- (“M1”) macrophages. MAA beads biased macrophages towards a M2
polarization state; noted by a decrease in M1 macrophages and an increase in M2 macrophages,
compared to MM beads. (E, F) Distribution of polarized macrophages: normalized number (E)
and frequency (F) of macrophages that were MHCII-CD206+, MHCII+CD206+,
MHCII+CD206-, and MHCII-CD206-. n = 3-4.
The polarization bias was reflected in the representative dot plots for MAA vs MM beads (Fig.
7A, B). Treatment with MAA beads led to significantly more MHCII-CD206+ (M2)
macrophages and decreased MHCII+CD206- (M1) macrophages, relative to MM beads at day 7
21
(Fig. 7C). On the contrary, treatment with MM beads had the opposite effect, with significantly
more M1 and fewer M2 macrophages relative to MAA beads at day 7 (Fig. 7D). In PEG-treated
animals, the number of M1 and M2 macrophages remained steady over the three time points, as
expected. By day 7, the majority of macrophages in MM-treated mice were double-positive
(MHCII+CD206+) or double-negative (MHCII-CD206-) (Fig. 7E, F). Interestingly, the number
of double positive macrophages increased progressively from day 1 to day 7 in MM- but not
MAA-treated animals (Fig. 7E). Conversely, in MAA-treated mice, macrophages were
consistently MHCII-CD206+ (M2) from day 3 onwards to day 7 (Fig. 7E, F). In MM-treated
mice, the progressive increase in MHCII+CD206- (M1) macrophages suggested that
macrophages may have been fusing to form foreign body giant cells [38]. Consistent with this
observation, BMDM-induced fusion using IL-4 in vitro formed large cells that resembled foreign
body giant cells (FBGCs) (Appendix, S7); these cells were MHCII+ and MHCII+CD206+,
suggesting that FBGCs expressed MHCII.
3.4 Interrogating biomaterial-cell interactions in intact tissues
Next, the spatial orientation of M2 macrophages relative to MAA beads and blood vessels was
investigated. Alexa 647-conjugated dextran (70kDa), the ligand for the CD206 receptor[58], was
injected into MAA- or MM- treated animals 30 min prior to injection of the Alexa 555-
conjugated lectin (GSL1) to visualize cells that expressed the CD206 scavenger receptor and to
label blood vessels, respectively. A flow cytometry strategy was devised to evaluate the cells that
associated with the lectin (Appendix S8). Two gating strategies were employed: 1) Gating first
for macrophages, then dextran positive cells, and 2) gating first for dextran positive cells, then
dextran positive macrophages (Fig. 8A, Appendix S8). Both gating strategies produced
comparable results. Approximately 90% of dextran+ cells were macrophages
(F4/80+CD11b+Ly6G-CD45+ cells) (Fig. 8B). Of the dextran+ macrophages, approximately
90% were CD206+, while approximately 40% were MHCII+ (Fig. 8C). Thus, of all cells that
were associated with dextran, about 90% × 90% = 88% were CD206+ while 90% × 40% = 36%
were MHCII+. Hence, dextran+ cells were considered CD206+ (M2-like) macrophages. There
was no difference in the percentage of dextran+ cells between MAA- or MM- treated animals
(Fig. 8C). Unlike the previous definition of M1 (MHCII+CD206-) or M2 (MHCII-CD206+)
macrophages, the dextran label was unable to distinguish macrophages that were MHCII-
CD206+ or MHCII+CD206+.
22
3.4.1 Effect of MAA beads on CD206 expression in surrounding macrophages
Dextran+ macrophages were localized to the immediate vicinity (<200 μm) of vessels (Fig. 8D)
regardless of treatment with MAA or MM beads. However, in MAA-treated animals, dextran+
macrophages were found in the immediate vicinity (<200 μm) of MAA beads, even in the
absence of vessels (Fig. 8D, bottom). In MM-treated animals, dextran+ macrophages were
located further away from MM beads and in areas with lectin staining (vessels). In agreement
with previous observations (Fig. 4, 5), a dense layer of cells (presumably F4/80+ based on the
histological analyses, Fig. 5) were found surrounding MM, but not MAA beads.
Random slices were selected from each image stack and the density of dextran+ macrophages
were quantified (Fig. 8E). As expected, the number dextran+ macrophages surrounding MAA
beads was higher relative to MM beads. Notably, the cells adhered to MM beads were not
dextran+, suggesting that they were not M2 macrophages. Together, these results suggested that
MAA beads biased macrophages towards the M2 polarization state, supporting the flow
cytometry results. Additionally, these observations highlighted the potential of CLARITY to be
used for the direct interrogation of cell-biomaterial interactions in explanted tissues.
23
Fig. 8. More CD206+ macrophages are found in the vicinity of MAA beads relative to MM
beads. (A) Representative flow cytometry gating strategy to determine dextran uptake by
macrophages (dextran 70 kDa = dex70). Data from MM-treated mice shown. (B) Frequency of
all dextran+ cells that were also macrophages (F4/80+CD11b+Ly6G-CD45+). (C) Frequency of
dextran+ macrophages that were CD206+ or MHCII+. Approximately 90% of all dextran+ cells
were macrophages, regardless of treatment (MAA vs. MM) and ~90% dextran+ macrophages
were CD206+ (vs. ~40% MHCII+). No differences were noted between MAA or MM explants;
n = 2. (D) Representative slices of tissues explanted from animals treated with MM beads (top)
or MAA beads (bottom). The arrows indicate the cells of interest. (E) Number of dextran+ cells
in the vicinity of MAA or MM beads. The image slices ranged from 200 to 800 μm into the
tissue. n = 2. Scale bar = 200 μm.
24
Discussion This chapter investigated the inflammatory cell response to methacrylic acid-containing beads
and showed that MAA beads promoted vessel formation. Moreover, treatment with MAA beads
promoted what we propose was an “alternative foreign body response”.
4.1 Effect of MAA beads on vessel formation
Previous studies in diabetic mice (BKS.Cg-m+/+ Leprdb/J mice, db/db) showed that MAA beads
increased vascularization in cutaneous wounds[16,49]. Here, subcutaneous injection of MAA
beads increased vessel density in non-diabetic mice relative to control MM beads (Fig. 4),
highlighting the vascular potency of MAA beads even in the absence of the physiological need
during diabetic wound healing. MAA beads nearly doubled the number of vessels at day 7
(~90% increase) (Fig. 4B). Only a few other synthetic biomaterials improve vascularization, to a
similar or frequently lesser degree[42,48,60].
To investigate the perfusability of newly formed vessels following treatment with MAA beads,
the explants containing beads (MAA and MM) were processed using a modified CLARITY
protocol. During CLARITY processing, light-scattering fatty lipids were removed while
proteinaceous structures and morphology were retained, enabling deep imaging and 3D
visualization of these fragile tissues[56,59]. Alexa 647-GSL1 (via tail vein injection; GSL-1 is a
lectin specific to mouse endothelial cells) staining was only observed around MAA and not MM
beads (Fig. 4C) indicating that the MAA-induced vessels were perfusable.
4.2 Effect of MAA beads on the inflammatory cell infiltrate
Treatment with MAA beads did not alter the number of F4/80+ macrophages in its vicinity
relative to control MM beads; however, the distribution of macrophages was different (Fig. 5A).
Flow cytometry analysis revealed that MAA beads altered the inflammatory cell landscape
relative to controls (Fig. 6). As expected, the presence of MAA beads resulted in more CD45+
leukocytes relative to the PEG vehicle control (Fig. 6A, Appendix S4). A fourfold increase in
Ly6G+ neutrophils was evident at day 1 in mice injected with MAA beads relative to both
controls (Fig. 6C). The link between MAA beads and neutrophil infiltration is not well
understood but may have been a result of protein (e.g., complement) adsorption differences (to
be discussed in Chapter 2). The significance of the increase in CD11c+ dendritic cells
25
(Appendix S4) is unclear. One caveat with the data was that the total number of cells was
determined by flow cytometry, with calibration beads used to determine the ratio between the
number of events and the number of cells. Cell numbers were further normalized by the mass of
the explants, recognizing that the volume of tissue that was digested varied to a small extent
from sample to sample. Although not statistically significant, higher explant masses and cell
numbers were recovered from MAA-treated animals. The reported numbers were reasonable
estimates of cell numbers, recognizing that we were interested in differences in inflammatory
cell infiltration over the course of the study. The normalization protocol may account for the
apparent increase in CD45- cells seen with PEG at day 1 (Fig. 6B). Explant masses and total cell
numbers were low with the vehicle-only controls (Appendix S3) so that after normalization, the
normalized numbers were artificially high. Following PEG treatment, the numbers of CD45+
leukocytes and CD45- non-leukocytes were unchanged from day 1 to day 7, as expected.
MAA beads increased the number of macrophages relative to MM beads at day 7 (Fig. 6D),
consistent with past observations of higher expression of TNFα and IL1β genes in diabetic
wounds at the same time point[49].The increase with histological analysis was not statistically
significant (Fig. 5B) presumably because of different regions of interest or a higher sensitivity of
flow cytometry to identify cells with lower levels of F4/80 expression. Although the analyses did
not distinguish between tissue-resident and bone-marrow derived macrophages, the mean
fluorescent intensity of F4/80 increased from day 1 to 7 for MAA and MM-treated animals (Fig.
6E), suggesting that macrophages were being recruited to the injection site, where they matured
over time. Macrophage maturation is associated with the expression of markers that are not
associated with blood-derived monocytes and changes in their transcriptome and proteome that
lead to fully-differentiated, tissue-resident cells[29,61].
In contrast to MAA beads, control MM beads were surrounded by a thick layer of F4/80+ cells
(i.e., macrophages) (Fig 5A); a common observation with implanted biomaterials (reviewed in
[5,6]). Similarly, a thick layer of cells was observed in Masson’s trichrome and CLARITY-
processed images around MM but not MAA beads (Fig. 4A, C). At day 7, MM-treated animals
had a higher density of CD45- cells (Fig. 6B), a majority of which were believed to be
fibroblasts. Overall, the distribution of F4/80 staining, the presence of a thick layer of
macrophages and a higher number of potentially fibroblasts suggested an increased fibrotic
response to control MM beads[41,62], a feature of a conventional foreign body
26
response[30,63,64]. On the other hand, the vascular regenerative MAA beads lacked a thick
layer of cells (Fig. 4A, C) and maintained low levels of CD45- cells (Fig. 6B); these are
indicative of what we have termed as an “alternative foreign body response”.
4.3 Effect of MAA beads on macrophage polarization
Macrophage phenotype varies depending on the conditions that have led to their
activation[32,65]; the M1/M2 distinction is an in vitro artifact and does not accurately reflect the
state of macrophages in vivo[32,66]. However, it is convenient to use the “M1” and “M2”
distinction as a simplification of the spectrum of polarization states. The importance of
macrophages has been readily tested; several groups have shown that elimination of
macrophages (via clodronate liposomes) detrimentally affects vessel formation[67] and that
addition of macrophages promotes neovascularization[68,69]. Despite the literature’s
considerable emphasis on the importance of M2 macrophages for vascularization, the extent of
their contribution remains unclear; the FBGC/ fibrotic qualities of M2 macrophages are often
ignored. While improved vascularization has been correlated with increased numbers of M2
macrophages[39], exogenous administration of M2 macrophages 1-3 days post-injury failed to
improve vascularization in a cutaneous wound model[70], although this may have reflected
changes that occur in pre-polarized macrophages upon implantation.
Macrophages, regardless of their polarization, have been shown to contribute to
vascularization[41]. Classically-activated, “M1” macrophages have a role in initiating vessel
formation[41] and alternatively-activated, “M2” macrophages that arise later in the foreign body
response are involved in promoting vessel maturation[41,71,72]. We hypothesized that MAA
beads orchestrated macrophage polarization towards the M2 state, consistent with the increased
vascularization. Treatment with MAA beads induced a M2 macrophage polarization bias (Fig.
7A, B). Flow cytometry analysis enabled quantification of macrophages that were CD206+, and
allowed for discrimination between those cells that were MHCII+ or MHCII-. Recognizing that
macrophage polarization is a complex spectrum, we designated MHCII+CD206- cells as M1
cells and MHCII-CD206+ cells as M2; double positive cells were also counted, although these
were neither M1 nor M2. At day 7, MAA beads increased the density of M2 cells fourfold
compared to MM beads (Fig. 7C), while MM beads induced a nearly nine-fold increase in M1
macrophages relative to MAA beads (Fig. 7D). Treatment with controls (MM beads) elicited a
27
more inflammatory macrophage response, with higher numbers of M1 and double positive
MHCII+CD206+ macrophages by day 7 (Fig. 7E, F). The latter are presumed to be cells in
transition from the initial inflammatory M1 cells to the later M2 cells, but additional research is
required to understand the role of these “hybrid” macrophages in vascularization. We think that
the increased numbers of M2 macrophages earlier in the host response promoted more vessel
maturation, resulting in a denser and perfusable vascular network.
The thick layer of cells (revealed to be F4/80+ macrophages, Fig. 5A) around MM but not MAA
beads, combined with the progressive increase in MHCII+CD206- (M1) macrophages in MM-
treated animals suggested the formation of FBGCs. Formation of large, MHCII+ cells that
resembled foreign body giant cells were noted in vitro in BMDM cultured with IL-4 (Appendix,
S7). Others have also noted the increased expression of MHCII in FBGCs[73]. MHCII and
CD206 are used as M1/M2 markers and do not accurate reflect the exact phenotype of the
labelled cells. Thus, the increase in MHCII+ and MHCII+CD206+ macrophages observed in
MM-treated animals may be an artifact of the markers used and may not be representative of M1
or hybrid macrophages.
4.4 Insights into MAA-mediated macrophage polarization using CLARITY
Macrophage polarization was further investigated using CLARITY to interrogate the spatial
distribution of macrophages in the context of MAA beads. Flow cytometry analysis revealed that
dextran (via tail vein injection) was associated with primarily CD206+ macrophages (~88% of
all dextran+ macrophages also expressed CD206+) (Fig. 8). Treatment with both MAA or MM
beads showed similar percentages, indicating that this observation was consistent between
treatment groups (Fig. 8C). Dextran+ macrophages were closely associated with vessels, but also
observed around MAA beads, in the presence and absence of vessels (Fig. 8D). In MM-treated
animals, the majority of the dextran+ macrophages were observed around blood vessels. The
increased expression of CD206 in cells surrounding MAA beads but not MM beads (Fig. 8E)
suggests that MAA beads may be interacting with macrophages and influencing their
polarization state directly. This data further supported our flow cytometry data and suggested
that MAA may be directly or indirectly (via various signaling pathways or neutrophils)
influencing M2 macrophage polarization.
28
Histological analysis on CD206-stained MAA and MM tissue sections in the same, albeit
transgenic mouse model produced a similar trend; although not statistically significant, MAA
treatment promoted more CD206+ cells in the vicinity of the beads at day 7 [15]. However, the
average CD206+ cell density was notably higher in the histological analyses relative to the
dextran-CLARITY method (Fig. 8E), which may have been a result of non-specific binding of
the CD206 antibody or the quantification strategy with histology. While it is unclear if the use of
dextran afforded higher specificity, what is clear is that the CLARITY protocol enabled the
direct interrogation of cells (i.e., dextran+ cells) whose labeling could potentially be validated
and quantified via flow cytometry simultaneously (from two tissue explants or one explant
divided in half). As the CLARITY technology advances and becomes more reliable and scalable
and strategies of labeling specific cells becomes available[74], it has the potential to rival
conventional histological protocols.
MAA beads promoted vascularization when implanted subcutaneously and the present data
suggests that M2 macrophages are one aspect of MAA’s vascular regenerative mechanism.
However, the extent of the contribution of M2 macrophages remains to be elucidated.
Macrophage depletion studies involving clodronate-liposomes conducted in a different system
(e.g., modules, grafts, etc.) revealed that macrophages are essential to vascularization; their
depletion leads to significantly reduced vessel formation[67]. It is necessary to conduct a
variation of a M2 macrophage knockdown to sufficiently demonstrate the importance of this
macrophage phenotype in the context of MAA. Yet, it may be difficult to design knockdown
studies that inhibit MAA-mediated M2 macrophage polarization, without adversely effecting
physiological vascularization as a whole. For example, a “M2 knockdown” may affect total
macrophage numbers, which would affect vessel formation[35,67]. Moreover, as macrophages
are polarized directly at the site of inflammation, it may be difficult to selectively knockdown
M2 macrophages, without adversely affecting M1 and other macrophage polarization states. To
this end, we propose to use the same subcutaneous injection model in Balb/c and C57BL/6 mice.
Balb/c mice are known to have a M2- biased inflammatory response while C57BL/6 have a M1-
biased inflammatory response; CD1 mice are in the middle of this spectrum and do not have a
M1-biased or M2-biased response [75]. Such experiments could shed light on the importance of
MAA-mediated macrophage polarization without affecting physiological functions.
29
While it is evident that MAA beads induced a bias in macrophage polarization towards M2, it is
unclear why this happens. Several interconnected mechanisms are likely involved and Chapter 2
aims to clarify these mechanisms. Knowledge of these mechanisms would translate to smarter
biomaterial designs for mediating vascularization and M2 macrophage polarization.
30
Conclusion
This chapter demonstrated that MAA beads promoted the formation of a denser and perfusable
network of blood vessels after subcutaneous injection, relative to control MM beads. Aim 1
revealed that the higher vessel density was accompanied by changes in inflammatory cell
infiltration (i.e., more neutrophils at day 1 and macrophages at day 7) and a macrophage
polarization bias towards the M2 state. Aim 2 explored this polarization bias further using
CLARITY, and revealed more M2-like macrophages in the immediate vicinity of MAA beads.
Together, these results suggest that MAA promoted an “alternative host response” (i.e., a foreign
body response distinct from the standard fibrosis) that is involved in MAA’s beneficial vascular
regenerative effect.
Chapter 2 Role of complement activation in MAA-mediated macrophage
polarization
Introduction
An altered inflammatory response involving M2 macrophage polarization is one element of a
complex network of pathways activated by MAA-based biomaterials to effect vascular
regeneration. The underlying mechanisms behind this polarization bias are the focus of this
chapter.
1.1 Protein-biomaterial interactions in the host response
The interactions between blood and a material is intimately associated with the inflammatory and
healing responses[76]. The inflammatory response is initiated by damaged tissues, but it is
modulated by the chemicals released from cells and those present in the plasma[5]. Within
milliseconds of contact between biomaterial and blood, a mixture of clotting factors (reviewed in
[21]) and complement proteins (reviewed in [77]) adsorb to the surface of the biomaterial,
initiating thrombosis, complement activation, and other blood-derived cascades[78]. The
acquisition of the layer of adsorbed proteins is an inevitable consequence of biomaterial
implantation[79]. One immediate and key mediator of this in vivo environment is the
complement system.
Complement is heralded as a major problem of biomaterial implantation; its activation promotes
adverse side-effects leading to poor biomaterial biocompatibility[6,63]. Blood-derived
complement proteins are the first arm of the innate immune system and are able to recognize
pathogens and foreign materials (reviewed in [80]). Upon contact with a biomaterial,
complement may be activated via three distinct pathways: 1) the classical pathway, 2) the lectin
pathway, and 3) the alternative pathway. The initiation of these pathways leads to the activation
of a cascade of proteases that converges at the level of C3 convertase, which mediates the
production of the anaphylatoxins C3a and later C5a, and ultimately the assembly of a terminal
complement complex (TCC) on a cellular surface, which forms pores in the cellular
membranes[80]. The anaphylatoxins propagate the inflammatory response by recruiting and
activating phagocytic cells (e.g., neutrophils and monocytes), triggering mast cell degranulation,
32
increasing vascular permeability, and inducing oxidative stress[6,19,63]. In the classical
pathway, adsorbed immunoglobulins (primarily IgG) are tagged by C1q, leading to the formation
of the C1 complex, which, via a cascade of proteolytic cleavages mediates the formation of C3
convertase. C3 can also adsorb directly on biomaterial surfaces and adopt an alternative
structure, initiating the alternative complement pathway[81]. Non-specifically adsorbed
carbohydrates and glycoproteins may also be recognized by the mannose binding lectin complex
(MBL), initiating the lectin pathway[80]. Dysregulated complement activation can lead to a
number of diseases, including kidney diseases and autoimmune diseases[82,83].
The potent effector functions of complement activation products have the potential to harm the
host. Thus, complement activation is a tightly-regulated process to prevent non-specific injury.
Initiators and inhibitors exist to regulate the location and activity of the complement system[81].
The C1 inhibitor (C1INH) binds to the C1 complex, as well as components of the lectin pathway,
inhibiting the activation of both the classical and lectin pathways[81]. Various plasma-derived
binding proteins and factors, such as C4 binding protein (C4BP), decay-accelerating factor
(DAF), Factor H, and others, accelerate the dissociation of the various complement proteins to
prevent hyperactivity. For example, DAF accelerates the dissociation of both the C3 and C5
complexes[81]. Various small-molecule drugs have been shown to mimic the function of some
of these regulators (Fig. 9). Pentamidine is a serine protease that inhibits C1s, analogous to
C1INH, inhibiting complement activation at the C1 level[84]. Aurin tricarboxylic acid (ATA)
inhibits the cleavage of C3b-Factor B to the active C3 convertase (C3b-Factor Bb) as well as the
attachment of C9 to C5b678; downstream of all three complement pathways.
33
Fig. 9. Drug-induced inhibition of complement activation. Pentamidine inhibits the classical
pathway. Aurin tricarboxylic acid (ATA) inhibits complement activation at the C3 and C5b-9
levels. A graphical depiction of the proteins adsorbed onto MAA beads is shown at the top of the
figure. Adapted from web.
1.2 Mechanisms of macrophage recruitment and polarization
It is traditionally thought that neutrophils promote the recruitment of monocytes to the site of
inflammation via the release of inflammatory markers (e.g., IL-1β, TNF-α, etc.); however, it has
since been demonstrated that monocyte recruitment may be independent of neutrophils[61].
Circulating monocytes respond to similar inflammatory signals as neutrophils and eventually
make their way to the site of inflammation. Neutrophils are primed to arrive faster due to
probability; there are significantly higher numbers of neutrophils circulating in the blood[22].
Therefore, complement activation products, such as C3a, are recognized by monocytes and
promote their recruitment to the site of inflammation[85]. Once differentiated into macrophages,
the inflammatory milieu of the implant site polarizes macrophage towards the M1 state, where
they play important roles in propagating inflammation and initiating vascularization. The
mechanisms that facilitate the transition from M1 to M2 macrophages are still not fully
understood; several mechanisms are proposed here.
34
1.2.1 Neutrophils
Neutrophils become apoptotic several hours after tissue infiltration. Apoptotic neutrophils have
been long known to be engulfed by macrophages[25]. More recently, it was shown that the
phagocytosis of apoptotic neutrophils promoted macrophages to adopt an M2 phenotype, seen by
an increase in the expression of anti-inflammatory genes [86] and prostaglandin E2, a key
mediator of vascularization[87]. Others also reported that the phagocytosis of apoptotic
neutrophils required macrophages to adopt a M2-like phenotype[88].
1.2.2 Complement proteins
Complement activation stimulates neutrophil infiltration, but their role in macrophage
polarization is not well defined. C1q, one of the key initiators of the classical pathway, was
shown to directly modulate macrophage polarization towards an alternatively activated “M2-
like” phenotype[89]. C1q was reported to interact with macrophages to stimulate the
upregulation of IL-10 and IL-33, polarizing macrophages towards the M2 phenotype,
accompanied by an upregulation in the apoptotic cell engulfment and a downregulation of the
NLRP3 inflammasome pathway[90]. On the contrary, others reported that C3-deficient mice
displayed increased neovascularization, albeit in a model of retinopathy, and that this effect was
mediated by macrophages[91]. Macrophages stimulated with C5a adopted an angiogenesis-
inhibitory phenotype, characterized by upregulated secretion of soluble VEGFR1, which
quenches VEGF, a potent initiator of vessel formation[91]. The disparity shown with these
examples highlight the diversity of mechanisms employed by complement proteins to influence
macrophage polarization.
1.2.3 IGF signaling pathway
IGF-1, a potent inducer of tissue regeneration, has been implicated in macrophage
polarization[92]. Expression of IGF-1 was increased in wounded tissues and
monocytes/macrophages were shown to be an initial source of IGF-1. Moreover, clonal deletion
of IGF-1 in specifically myeloid-derived cells impaired accumulation of CD206+ M2
macrophages [93]. Additionally, M(IL-4), “M2” macrophages were shown to upregulate IGF-1
expression [11] and macrophages from IGF-1 KO animals failed to generate a pro-healing
phenotype associated with M2 macrophage polarization – suggesting a role for IGF-1 in
promoting M2 macrophage polarization [93].
35
1.3 Biomaterial strategies for mediating macrophage polarization
Biomaterials are being used to effect M2 macrophage polarization. Mokarram et al developed
polymeric nerve guide channels that enabled the sustained release of IFNγ or IL-4 to promote
macrophage polarization towards the “M1” or “M2” state respectively, in a peripheral repair
model[94]. Polymers that released IL-4, but not IFNγ improved nerve repair while promoting a
higher ratio of CD206+/CCR7+ macrophages, indicating macrophage polarization towards the
M2 state. They attributed the increased nerve healing to the induction of M2 polarized
macrophages and the subsequent recruitment of Schwann cells. Others have attempted to take
this idea further in other models. Spiller et al developed a sequential-release strategy to deliver a
sequence of signals to enhance 1) M1 macrophage polarization, then 2) M2 macrophage
polarization[95]. Their bone scaffolds released IFNγ rapidly, then IL-4 over time to modulate the
shift from M1-to-M2 macrophages to promote vascularization. Interestingly, increased vessel
formation was only observed in animals implanted with scaffolds containing IFNγ and not
scaffolds containing IL-4 or the combination of IFNγ and IL-4. Although it is debatable if their
technique truly incorporates sequential IFNγ/IL-4 release, their observations highlight the
complexity of macrophage polarization. It is unclear why in one case IL-4 alone is able to
promote healing while in the other IFNγ alone was sufficient. Macrophage polarization is a
natural process of healing; M1 macrophage will inevitably dominate the early inflammatory host
response and M2 macrophages the late, healing response[28].
1.4 Complement modulating effects of MAA
MAA beads incubated with human serum showed preferential adsorption of various complement
proteins relative to MM beads (Wells, LA et al, Biomaterials, submitted). Among the adsorbed
proteins was the complement initiator C1q, and various inhibitor factors, such as factor H. The
presence of C4 and C3 suggested that complement was being activated on the surface of MAA
and MM beads. Interestingly, MAA beads incubated with human serum produced lower amounts
of C3a and C5b-9, suggesting that MAA beads inhibited complement activation. Protein
deposition on biomaterials is more complicated in vivo, involving the contribution of several
other blood-derived pathways which could alter the adsorbed layer of proteins over time[76].
The increased neutrophil infiltration following subcutaneous injection of MAA beads led us to
36
believe that MAA may indeed be activating complement and producing the anaphylatoxins C3a
and C5a to recruit more neutrophils and monocytes to the site of inflammation[15,96,97].
We sought to validate the in vitro findings and developed our hypothesis around the ideas that 1)
complement activation promotes neutrophil infiltration and 2) a less inflammatory response
involving lower numbers of apoptotic neutrophils reduces M2 macrophage polarization. We
hypothesized that inhibiting complement activation would 1) reduce neutrophil and monocyte
recruitment, 2) eliminate the M2 polarization bias induced by MAA, resulting in 3) lower vessel
densities. Our hypothesis aimed to link the events that occur immediate after biomaterial
implantation (i.e., complement protein adsorption) to the alternative host response and
vascularization response induced by MAA beads.
To investigate the role of complement in MAA-mediated macrophage polarization and
vascularization, we modified the subcutaneous injection model: CD1 mice were administered
either pentamidine or Aurin tricarboxylic acid (ATA) to inhibit complement activation at the C1
or C3 levels, respectively prior to the injection of MAA beads. Analogous to the previous study,
at day 1, 3, and 7 post-injection, the bead explants were processed for immunohistochemistry
and flow cytometry for the estimated number of cells and their polarization state (i.e.,
macrophages). Inhibition of complement activation eliminated the enhanced neutrophil
recruitment effect of MAA and reduced macrophage recruitment and the M2 macrophage
polarization bias at day 3; however, no notable changes in MAA-mediated vascularization were
observed.
37
1.5 Objectives
This chapter explores the role of complement activation on the MAA-mediated alternative host
response, with a focus on macrophage polarization, and vascularization.
Aim 2: Clarify the role of complement activation in MAA-mediated macrophage polarization
and vessel formation.
Hypothesis: Inhibition of complement activation reduces the number of infiltrating neutrophils
and monocytes and negates the effect of MAA beads on M2 macrophage polarization, leading to
lower numbers of CD31+ vessels.
38
Methods
2.1 Preparation of poly(methacrylic acid-co-isodecyl acrylate) films
Poly(methacrylic acid-co-isodecyl acrylate) (MAA-co-IDA) films or MAA films were composed
of 45 mol% methacrylic acid (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada), 1 mol%
ethylene glycol dimethacrylate (Sigma-Aldrich Canada Ltd.) and 54 mol% IDA (Sigma-Aldrich
Canada Ltd.). These were used for some in vitro studies.
MAA films were synthesized by suspension polymerization as previously described[50] and
were dissolved in THF immediate prior to coating glass cover slips. The methacrylic acid content
of the films was confirmed by titration. Control poly(methyl methacrylate) (MM-co-IDA) films
or MM films were synthesized in a similar fashion to MAA films. MAA and MM films, post-
coating, were washed in 95% ethanol twice and then rinsed twice in sterile PBS prior to use in
vitro. Analysis with a limulus amebocyte lysate (LAL) pyrochrome endotoxin test kit (Cape Cod
Inc., Falmouth, MA) indicated that beads contained <0.25 EU/100 mg.
2.2 Isolation, culture, and characterization of bone marrow-derived monocytes
Bone marrow-derived macrophages (BMDM) were harvested from CD1 mice using
conventional techniques[11,45]. Briefly, hind limbs were removed from the hip joint and placed
in PBS on ice. Muscle was removed from the femur and tibia, washed and rinsed in RPMI 1640
and bone marrow was obtained by flushing the bone marrow contents using a 25G needle. The
acquired bone marrow cells and debris were filtered, washed, and cultured in RPMI 1640
supplemented with 10% heat-inactivated FBS, 1% Penn/Strep, and 20 ng/mL of M-CSF
(Invitrogen) on non-tissue culture-treated polystyrene plates for 5-7 days. Media was changed at
day 3 to remove non-adherent, non-macrophage cells. Macrophages (adherent) were
differentially polarized using 20 ng/mL IFNγ (Invitrogen) and 20 ng/mL IL-4 (Invitrogen) for 48
hours. Polarization was confirmed using CD11b, F4/80, along with either CD206 for
alternatively- activated (M2) macrophages or CD86 and MHCII for classically- activated (M1)
macrophages (Appendix, S9).
39
2.3 Macrophage stimulation by biomaterials in vitro
Cultured bone marrow-derived macrophages (BMDM) were stimulated with MAA (0.3g/cm2) or
MM (0.9g/cm2) beads or MAA-co-IDA/MM-co-IDA films for 24-72 hours, in the presence, or
absence of whole mouse CD1 blood (0.1% to 1% v/v, remainder RPMI 1640 medium + 10%
FBS + 1% P/S, minus M-CSF). MAA beads and films were equilibrated to pH 7.4 prior to
mixing with whole blood. The BMDM were treated in one of three ways: 1) Beads (2.85 mg
MAA or 8.55 mg MM), blood, and culture media mixture (totaling 1 mL) was directly
transferred into a 6 well plate containing adherent BMDM. 2) MAA-co-IDA or MM-co-IDA
films were added directly onto BMDM seeded onto 0.4 μm cell inserts and placed into 6 well
plates. 3) Beads, blood, and culture media (totaling 500 μL) were transferred onto 48 well plates
containing adherent BMDM.
After 24-72h of incubation, the beads or films were removed and the cells were washed 3 times
with PBS. For flow cytometry analysis, cells were harvested from the plate/cell insert,
resuspended in PBS supplemented with 0.5% BSA and 2 mM EDTA, and stained with F4/80,
MHCII and CD206. In other situations, involving 48 well plates, the BMDM were fixed, blocked
and stained with MHCII-PerCPe710, CD206-PE, and DAPI directly on the plate. The
fluorescence intensity of each well was acquired using a fluorescence plate reader (Tecan
M200Pro) (Appendix, S9).
2.4 CH50 type hemolysis assays
Mouse serum was prepared by collecting 20 μL of blood from the tail vein in normal tubes,
followed by fibrinization for 2h at 37°C, then centrifugation at 3000×g for 5 min. For hemolysis
assays, a CH50 type system was employed, as previously described[98,99]. The supernatant was
collected and diluted by adding 100 μL of complement buffer (20 mM HEPES, 0.5 mM MgCl2,
0.15 mM CaCl2, 141 mM NaCl and 0.1% gelatin). Separately, sheep red blood cells (QuadFive)
were washed 3 times in complement buffer followed by centrifugation at 3000×g for 5 min, until
no red color was observed in the supernatants. The RBCs were diluted to 5 × 107 cells/mL and
incubated with complement buffer containing 100 μg/mL of hemolysin (Rabbit Anti-Sheep IgGs
and IgM, CedarLane) to sensitize. The CH50 assay was performed using a reaction mixture of 50
μL sensitized RBCs and 50 μL of mouse serum, diluted serially 1- to 16- fold. The mixture was
incubated at 37°C for 30 min. The supernatants (80 μL) were collected and transferred to a 96-
40
well plate (Fischer) and the optical density (OD) at 414 nm was read with a plate reader (Tecan).
As a positive control, the RBCs were 100% lysed with water, and as a negative control, no serum
was added to the incubate. Hemolysis (as a % of the maximum level of hemolysis) of was
calculated using the following formula: (ODsample-ODpos)/(ODpos-ODneg) × 100%, where ODpos
and ODneg are the positive and negative controls, respectively. Inhibition of complement
activation was compared using the IC50, expressed as a dilution fold instead of as a
concentration.
2.5 Complement drug inhibition study
Prior to subcutaneous implantation of MAA or MM beads, animals were treated with either: 1)
pentamidine (intraperitoneal injection; 4 mg/kg – 20 mg/kg, Sigma-Aldrich), which inhibited the
initiation of the classical pathway by blocking complement activation at the C1 level or 2) Aurin
tricarboxylic acid (ATA; orally; 500 mg per kg of mash, Aurin Biotech), which inhibited
complement activation at the C3 and C5b-9 levels (see Fig. 9).
Animals were treated with either MAA beads, MM beads, or MAA beads following complement
inhibition. Pentamidine (4 mg/kg – 20 mg/kg) was injected intraperitoneally starting one day
prior to subcutaneous injection of MAA beads. Animals were treated with pentamidine once per
day until sacrifice at days 1, 3, or 7. ATA (500 mg/kg) was mixed with mash and fed to animals
in the absence of chow starting one day prior to subcutaneous injection of MAA beads. The
ATA-supplemented mash was changed once per day to avoid mold formation. Each animal was
estimated to receive an approximate dose of 100 mg/kg of ATA per day. During the ATA
optimization process, ATA was administered by subcutaneous injection (up to 2.5 - 10 mg/kg).
Control animals were given either an equal volume IP injection of 0.85% saline or were fed an
ATA-free diet. CH50-type assays were performed prior to injection of MAA or MM beads to
validate inhibition of complement activation. All animal work was done with the approval of the
University of Toronto Animal Care Committee. Animals were housed under sterile conditions in
the University of Toronto’s Department of Comparative Medicine (AUP #20011359).
2.6 Tissue explant and digestion
Tissue explant and digestion were performed as described in Chapter 1, with the following
exceptions: 1) No PEG vehicle control was included, and 2) cell suspensions were stained with
41
live/dead stain, CD45, CD11b, F4/80, Ly6G, CD86, MHCII, and CD206. CD206 conjugated to
PE was used instead of CD206 conjugated to BV650. The markers CD11c and CD31 were not
used.
2.7 Analysis of cellular infiltrate in explanted tissues
A modified gating strategy was devised from the one reported in Chapter 1 (Appendix, S10).
After isolating live single cells, CD45 distinguished leukocytes from non-leukocytes.
Neutrophils were identified as Ly6G+. Macrophages were first identified as Ly6G-
F4/80+CD11b+ and then further characterized as MHCII+ CD206- (“M1”) and MHCII-CD206+
(“M2”). Cells were gated according to positive staining for each antibody using fluorescence
minus one (FMO) controls. Cell populations were expressed as either a percentage or as a
normalized value (estimated total number of cells divided by the weight of the explanted tissue).
2.8 Statistical Analyses
All values are reported as mean ± SEM, unless indicated otherwise. No statistical analysis was
conducted to compare complement-inhibited (n = 2) animals to non-complement-inhibited (n =
2-3) animals due to low n values. The reported changes in the host response only represent
trends.
42
Results
3.1 Investigating the mechanism of MAA-mediated macrophage polarization
In the subcutaneous injection model, it was noted that deliberate nicking of small vessels
enhanced the vascular regenerative effect of MAA beads (but not MM beads), suggesting a role
for whole blood or one of its components. Motivated by these results, an attempt was made to
study the effect of MAA on primary macrophages in vitro. As bone marrow-derived
macrophages (BMDMs) are a widely-accepted model used for macrophage polarization[32], we
opted to use BMDMs in our in vitro experiments.
3.1.1 In vitro analysis of BMDM treated with MAA beads and films
BMDMs treated with MAA beads or MM beads showed similar levels of CD206, CD86, or
MHCII, as analyzed by flow cytometry and fluorescence imaging (Appendix, S11). However,
when a small quantity of whole murine blood (0.5-1% v/v) was added with the MAA beads prior
to its addition to BMDM, the expression of CD206, but not MHCII increased; treatment with
MM beads had the opposite effect (Appendix, S11). Additionally, stimulation of BMDM with
MAA films (which enabled more material-BMDM contact relative to MAA beads) upregulated
arginase 1 (Arg1; an M2 marker) mRNA expression and did not significantly change iNOS (an
M1 marker) mRNA expression (Appendix, S12). These trends, albeit small, suggested that
MAA-containing materials were directly polarizing macrophages towards the M2 state.
Moreover, these data suggested a role for blood or its components (i.e., serum) in MAA-
mediated macrophage polarization.
3.2 Inhibition of serum-derived complement and its effect on MAA
Separately, we have seen that incubating beads with plasma or serum resulted in more
complement proteins (e.g., C1q, Factor H) adsorbed to MAA relative to MM beads yet
complement was activated to a lower degree with MAA beads (Wells, L.A. et al, Biomaterials,
submitted). The in vitro situation does not completely reflect the in vivo situation, which involves
a number of additional pathways that add to the complexity of complement activation[21,77].
We sought to shed light on the in vitro findings by investigating the effect of complement
43
activation in the context of MAA in vivo. Pentamidine and Aurin tricarboxylic acid (ATA) were
used to inhibit complement activation and the resulting effects on MAA-mediated macrophage
polarization and vascularization were evaluated. Pentamidine inhibited the proteolytic activity of
C1, preventing the cleavage of C4 and C2 and the classical arm of the complement pathway [84].
Aurin tricarboxylic acid (ATA) inhibited the cleavage of C3b- Factor B to the active C3
convertase (C3b-Factor Bb) as well as the attachment of C9 to C5b678 (See Fig. 9); inhibiting
complement activation further downstream than pentamidine, at the C3 and C5b-9 levels
[100,101].
3.2.1 Effect of complement inhibition on the vascular regenerative properties of MAA
The results of the CH50-type assays are shown in Fig. 10. Diluted serum (1- to 16- fold) was
incubated with sensitized red blood cells (RBCs). Serum from pentamidine-and ATA-treated
animals required less dilution to reach baseline (i.e., negative control) than control animals,
indicating that complement activation was inhibited. Indeed, one single intraperitoneal (IP)
administration of 20 mg/kg pentamidine inhibited complement-mediated red blood cell
hemolysis with minor changes in inhibition over 24h (Fig. 10A); the effect of IP administration
of 4 mg/kg pentamidine was shorter-lived (Appendix, S13). Subcutaneous injection of up to 10
mg/kg ATA inhibited hemolysis, but the effect was short-lived (Appendix, S13). Oral
administration (by mixing ATA with mash) of 500 mg/kg ATA sufficiently inhibited hemolysis
24h following administration (Fig. 10B). The fecal matter of ATA-fed animals was a distinct red
color relative to normal, non-treated animals (Fig. 10B), suggesting that some ATA was not
adsorbed by the body [100,102]. The sera of animals administered pentamidine or ATA had their
lowest IC50 values of 0.373- and 0.366-fold, respectively, indicating a ~5-fold enhanced
protection compared to control animals (IC50 1.891-fold) (Fig. 10C). Animals were administered
pentamidine or ATA once daily to ensure that complement was inhibited over the entire duration
of each experiment.
44
Fig. 10. Administration of pentamidine and ATA inhibited complement activation. (A, B)
Serum from complement-inhibited mice were compared to control mice administered 0.85%
saline or normal diets (indicated by Ctrl). CH50-type assay of CD1 mice treated with 20 mg/kg
pentamidine (A) or 500 mg/kg ATA (B). Data points represent means only. (C) Efficiency of
complement inhibition, presented as the IC50. The IC50 at 2h following 20mg/kg pentamidine
treatment was 0.373-fold and 24h following 500mg/kg ATA treatment was 0.366-fold, while the
control animals were 1.891-fold. A lower IC50 indicated better inhibition of complement
activation. Pooled data from n=3. The dotted line represents the IC50 of control animals not given
pentamidine or ATA.
Animals treated with pentamidine or ATA showed no dramatic difference in the number of
CD31+ vessels relative to controls (Fig. 11A, B). Similar vessel densities were noted in
pentamidine- or ATA- treated animals at day 3 (Fig. 11C) relative to animals treated with MAA
alone. A similar observation was noted in pentamidine-treated animals at day 7. The day 7 time-
point was not evaluated in ATA-treated animals. A thick layer of cells was not seen around
MAA beads, regardless of complement inhibition. Together, these data suggested that the
complement inhibition at the C1 or C3 levels did not dramatically affect MAA-mediated
vascularization.
45
Fig. 11. Inhibition of complement activation did not affect the vascular potency of MAA.
(A, B) Histology sections of animals treated with MAA (A-left) or MM beads (A-right) or MAA
with either 20 mg/kg pentamidine (B-left) or 500 mg/kg ATA (B-right) at day 3. Arrows indicate
examples of vessels. (C) Inhibition of complement activation did not dramatically affect vessel
densities at day 3 or day 7. Scale bar = 200 μm. Pentamidine or ATA-treated animals; n = 2.
MAA- and MM-treated animals, n = 2-3.
3.2.2 Effect of complement inhibition on MAA-mediated inflammatory cell infiltration
Administration of pentamidine and ATA prior to subcutaneous injection of MAA beads modified
the MAA-mediated alternative host response. Using the modified gating strategy outlined in
Appendix, S10, no difference was noted in the mass of the tissue explants, the estimated total
number of cells, and the normalized cell numbers (Appendix, S14). The PEG vehicle control
was removed. No difference was noted in the densities of CD45+ or CD45- cells in complement-
inhibited animals relative to animals treated with MAA beads alone (Fig. 12A, B). Although a
progressive increase in CD45- cells (i.e., non-leukocytes) in pentamidine-treated animals was
observed (Fig. 12B), analogous to MM-treated animals. The frequencies of CD45+ and CD45-
cells (as a percentage of live cells) followed similar trends (Appendix, S15).
46
Fig. 12. Complement inhibition eliminated MAA’s enhanced neutrophil recruitment effect.
(A-C) Number of CD45+ leukocytes (A), CD45- non-leukocytes (B), and
Ly6G+CD11b+CD45+ neutrophils (C) in animals treated with 20 mg/kg pentamidine or 500
mg/kg ATA or MAA or MM beads alone. Administration of pentamidine lowered the numbers
of neutrophils (C) at day 1. (D) Representative dot plot of Ly6G+ neutrophils compared between
MAA-treated animals and pentamidine-treated animals. (E) Number of F4/80+ Ly6G-
CD11b+CD45+ macrophages. Inhibition of complement activation decreased macrophage
numbers at day 3 and day 7. MAA- and MM-treated animals, n = 2-3. Pentamidine/ATA-treated
animals; n = 2.
Inhibition of complement activation dramatically reduced the estimated number of neutrophils
recruited to the site of MAA beads at day 1 (Fig. 12C); a prominent decrease in neutrophil
density was observed in the representative dot plots (Fig. 12D). Inhibition of complement
activation decreased the number of macrophages at day 3 relative to treatment with MAA or MM
beads alone (Fig. 12E). Additionally, in pentamidine-treated animals, fewer macrophages were
observed at day 7. Overall, these results indicated that inhibition of complement activation
modified the MAA-driven host response; resulting in lower levels of neutrophil and macrophage
infiltration.
3.2.3 Effect of complement inhibition on MAA-mediated M2 macrophage polarization
Next, flow cytometry was used to distinguish macrophages between the “M1 (MHCII+CD206-)”
and “M2 (MHCII-CD206+)” polarization states, as described previously. There were some small
47
differences in macrophages expressing MHCII and CD206 following complement inhibition,
with a general trend involving less CD206+ expression in complement-inhibited animals at day 3
(Appendix, S15).
Treatment with pentamidine or ATA altered MAA-mediated macrophage polarization at day 3
(Fig. 13). The shift in polarization was reflected in the representative dot plots for pentamidine
or ATA-treated animals, relative to controls (Fig. 13A-C). Animals treated with pentamidine or
ATA prior to subcutaneous injection of MAA beads had lower numbers and frequencies of
MHCII-CD206+ (M2) macrophages at day 3, relative to animals treated with MAA beads alone
(Fig. 13D). However, by day 7, the number of M2 macrophages in pentamidine-treated animals
was restored to levels comparable to animals treated with MAA beads alone (Fig. 13D).
Conversely, the number of MHCII+CD206- (M1) macrophages remained at levels comparable to
animals treated with MAA beads only for the duration of the experiments (Fig. 13E), although a
small spike in these M1 macrophages was noted in pentamidine-treated animals at day 3.
48
Fig. 13. Complement inhibition altered the MAA-mediated effects on M2 macrophage
polarization. (A-C) Representative dot plot of F4/80+ cells (macrophages) in mice treated with
MAA beads (A) or pentamidine (20 mg/kg) (B) and ATA (500 mg/kg) (C) prior to subcutaneous
injection of MAA beads at day 3. (D, E) The number and frequency of the individual single
positive, double positive, and double negative MHCII or CD206 macrophage populations in
mice treated with MAA beads, MM beads, MAA + pentamidine or MAA + ATA. (D)
Normalized number and frequency of MHCII-CD206+ (“M2”) macrophages. (E) Normalized
number and frequency of MHCII+CD206- (“M1”) macrophages. Inhibition of complement
activation eliminated the M2 macrophage polarization bias observed in animals treated with
MAA beads alone. (F, G) Distribution of polarized macrophages: normalized number (F) and
frequency (G) of macrophages that were MHCII-CD206+, MHCII+CD206+, MHCII+CD206-,
and MHCII-CD206-. MAA- and MM-treated animals, n = 2-3. Pentamidine/ATA-treated
animals; n = 2.
49
Although by day 7, pentamidine-treated animals showed lower numbers of macrophages relative
to MAA alone controls (Fig. 13F), the polarization of macrophages was similar to MAA-treated
animals (Fig. 13G). Interestingly, the number of double positive (MHCII+CD206+)
macrophages in pentamidine-treated animals increased progressively over time, mimicking that
of MM-treated animals (Fig. 13F, G). The population of double-positive macrophages was small
in ATA-treated animals; instead, the majority of macrophages were double-negative (MHCII-
CD206-) at day 3 (Fig. 13G). Overall, these results indicated that complement inhibition
eliminated the M2 macrophage polarization bias mediated by MAA beads at day 3, but not at
day 7.
50
Discussion This chapter aimed to connect the protein-adsorption events that occur immediately following
MAA biomaterial implantation to the later changes in the host response. Previous studies showed
that more complement proteins (e.g., C1q, factor H, C4) were adsorbed onto MAA beads relative
to MM beads, although in vitro experiments showed inhibition of complement activation (Wells,
L.A. et al, Biomaterials, submitted). Recognizing the more complex nature of the in vivo
situation, and motivated by in vitro findings suggesting the involvement of serum in MAA-
mediated macrophage polarization (Appendix, S11, S12), this study explored the role of
complement activation in the context of MAA in vivo. The increased deposition of C1q and C4
suggested activation of the classical pathway; thus, pentamidine was used to inhibit complement
activation at the C1 level. Aurin tricarboxylic acid (ATA) was used to inhibit complement
activation at the C3 level as other complement pathways (i.e., the alternative pathway) could
potentially compensate for inhibition at the C1 level. The sera of pentamidine- or ATA- treated
animals gave a maximum ~5- fold protection compared to control animals (based on IC50) (Fig.
10), as expected. Complement inhibition at the C1 and C3 stages altered the MAA-mediated
alternative host response and macrophage polarization, but did not affect vascularization.
4.1 Role of complement inhibition in MAA-mediated vascularization
Treatment with pentamidine or ATA prior to subcutaneous injection of MAA beads resulted in a
marginal decrease in CD31+ vessels relative to animals treated with MAA alone at day 3. At day
7, similar numbers of CD31+ vessels were noted in pentamidine-inhibited and control animals
(Fig. 11). C1qa-/- knockout mice (a knockout of subcomponent a of the larger C1q complex)
exhibit impaired vascularization following injury; C1q is thought to directly interact with
endothelial cells to effect vascularization[103]. In this C1qa-deficient model, a lack of C3 and C4
deposition indicates marginal classical complement pathway activation, suggesting that C1q
independently mediates vessel formation without complement activation. On the contrary, C3-/-
knockout mice exhibit enhanced neovascularization, albeit in a model of retinopathy [91]. These
studies suggest that aspects of MAA-mediated activation may occur independently of the host
response and directly via C1q and endothelial cells, for example. Additionally, complement-
deficient animal models (knockout or knockdown models) likely inhibit complement activation
51
better (lower IC50) than Pentamidine or ATA. Thus, in our study, incomplete complement
inhibition may have contributed to the lack of change in vessel densities.
Although complement inhibition reduced MAA-mediated M2 macrophage polarization (to be
discussed below), there was no correlation to vessel density. The marginal drop in vascularity in
pentamidine and ATA-treated animals at day 3 was likely an artifact of low n and could be
explained by the lower number of macrophages in complement-inhibited animals at day 3 (Fig.
12E). We did not investigate vessel maturity, which may have been a more specific metric for
functional M2 macrophages than vessel density. One important factor secreted by M2
macrophages is PDGF, which recruits pericytes to the blood vessels, facilitating vessel
maturation[72].
4.2 Role of complement inhibition in MAA-mediated alternative host response
Complement has been implicated as an important mediator of inflammatory cell infiltration (e.g.,
neutrophils and macrophages) [85,104]. Adsorption of complement proteins onto biomaterials
leads to the activation of the classical, lectin, or alternative complement pathways, resulting in
the production of the anaphylatoxins C3a and C5a, which in turn promotes neutrophil and
monocyte recruitment[5]. Interestingly, our previous data suggested that MAA beads had
complement-inhibiting properties in vitro (Wells, L.A. et al). However, the increased neutrophil
infiltration observed in our most recent dataset was not consistent with reduced complement
activation, although complement is not the only driver for neutrophil infiltration[15]. We sought
to clarify these observations and for the first time, connect the events that occur immediately
following biomaterial implantation (i.e., complement protein deposition) to the alternative host
response mediated by MAA.
Administration of pentamidine and ATA prior to subcutaneous injection of MAA beads modified
the MAA-mediated alternative host response. A dramatic drop in Ly6G+ neutrophils was
observed in complement-inhibited animals relative to MAA controls at day 1 (Fig. 12C, D).
Furthermore, a decrease in the number of macrophages was noted at day 3 in complement-
inhibited animals relative to MAA and MM beads (Fig. 12E). It was likely that complement
inhibition led to lower levels of C3a and C5a, promoting less neutrophil and monocyte
infiltration to the site of biomaterial injection. The small, but progressive, increase in CD45-
52
cells in pentamidine-treated animals may have indicated more fibroblast recruitment and a more
conventional host response (Fig. 12B).
4.3 Role of complement inhibition in MAA-mediated M2 macrophage polarization
As apoptotic neutrophils are engulfed by macrophages, a shift in macrophage polarization from
M1 to M2 is induced[28,88]. C1q, a component of the larger C1 complex and an initiator of the
classical pathway, has been implicated in macrophage polarization[89]. C1q and other members
of the opsonin family (e.g., MBL and adiponectin) downregulate inflammasome activation in
macrophages while upregulating the phagocytosis of apoptotic cells, consistent with the M2
polarization state. Thus, complement may directly and indirectly affect macrophage polarization
through C1q and apoptotic neutrophils, respectively. We hypothesized that the complement-
induced dampening of inflammatory cell infiltration would lead to fewer apoptotic neutrophils
and consequently less M2 macrophage polarization.
Indeed, complement inhibition reduced the MAA-mediated M2 macrophage polarization (Fig.
13A-C). A notable drop in the number and frequency of M2 (MHCII-CD206+) macrophages
was observed at day 3 in both pentamidine and ATA-treated animals; however, by day 7, this
effect was nullified; the number and frequency of M2 macrophages in pentamidine-treated
animals were comparable to animals treated with MAA alone (Fig. 13D). The number of M1
(MHCII+CD206-) macrophages in complement-inhibited animals followed a similar trend to
MAA only animals (Fig. 13E). A small spike in M1 macrophages was noted in pentamidine-
treated animals at day 3 but lowered to levels similar to animals treated with MAA only at day 7.
(Fig. 13E). Together with the restored M2 macrophage polarization in pentamidine-treated
animals at day 7, these data suggest that there may be other pathways compensating for the effect
of complement activation. Alternatively, it may suggest that the MAA-mediated M2 polarization
bias was delayed due to the less robust inflammatory response.
Indeed, MAA beads activated the sonic hedgehog (Shh) signaling pathway, as observed by the
upregulation of Shh and its receptor Ptch1 (patched 1) in surrounding cells in the same
subcutaneous injection model[15]. Shh expression was upregulated in MAA film-treated BMDM
in vitro, suggesting a link between Shh and M2 macrophage polarization. The expression of Shh
has been associated with increased macrophage infiltration and expression of M2-related genes,
53
such as IL-10, suggesting that Shh may act as a M2 polarization signal[105]. The insulin-like
growth factor (IGF) signaling pathway has also been implicated in healing and macrophage
polarization. Clonal deletion of IGF-1 in myeloid-derived cells impaired accumulation of
CD206+ M2 macrophages in wounded muscle; suggesting a role for IGF-1 in promoting M2
macrophage polarization[93]. Indeed, past genomic and phosphoproteomics screens identified
the IGF signaling pathway as a prominent pathway that was engaged following MAA
treatment[51,53]. More recently, BMDM stimulated with MAA films were shown to secrete
more IGF-1. Endothelial cells treated with MAA-stimulated BMDM conditioned medium
improved their proliferative and migratory capabilities; BMDM-derived IGF was revealed to be
a main mediator of this effect. In the same study, BMDM treated with MAA films adopted an
M2-like phenotype (Appendix, S12), further suggesting that IGF-1 modulates M2 macrophage
polarization. These alternative pathways of M2 macrophage polarization may have compensated
for the inhibition of complement activation.
In our inhibition experiments, an important control (MM + drug) was missing; it was unclear
whether the effect of complement inhibition was affecting MAA-mediated macrophage
polarization or simply macrophage polarization in general. This being said, since the addition of
pentamidine or ATA did not dramatically affect vascularization, it was unlikely that pentamidine
or ATA had adverse effects on physiological macrophage polarization and may have only
influenced MAA-mediated effects. Complement protein knockout models (e.g., C1qa-/- or C3-/-
mice) may inhibit complement activation better than pentamidine or ATA (and are likely more
specific), but there are challenges associated with these models. If lower vessel densities are seen
in C1qa-/- animals, it may be difficult to distinguish whether this phenomenon is a result of the
knockout affecting MAA-mediated effects or rather, physiological vascularization as a whole.
Since MAA-based biomaterials are thought to act as agonists of biological responses, it is
necessary to tease out which responses are the most important. The data from the previous
phosphoproteomics[53] and current mass spectrometry studies (Wells, L.A. et al, Biomaterials,
submitted) could be employed here. A correlation analysis could be employed to correlate the
proteins and ligands most readily adsorbed to MAA (Wells, L.A. et al, Biomaterials, submitted)
to the cellular signaling pathways that are activated [53], rather than evaluating each component
individually. This would provide a more rigorous method of identifying signaling pathways to
follow up on.
54
4.4 Insight into the mechanism of vascular regenerative MAA beads
The results described here and previously (reviewed in [7]) revealed that MAA-based
biomaterials elicit its regenerative properties by modulating several aspects of vascular biology,
including macrophage polarization. We think that upon interaction with tissues, MAA-based
biomaterials adsorb proteins, such as complement and numerous others (Wells, LA. et al,
Biomaterials, submitted), which then modulate phosphorylation pathways within minutes of
contact between the biomaterial and cells[53]. These interactions modulate the subsequent host
response; differential expression of mRNA and proteins promote an alternative foreign body
response, characterized by increased neutrophil infiltration and monocytes. This in turn results in
the activation of other signaling pathways (e.g., Shh[15], IGF signaling, etc.), neutrophil
apoptosis, and the modulation of cells involved in vascularization (i.e., macrophages and
endothelial cells). It is clear that macrophages and complement activation are two small pieces to
a much larger and complex network of events engaged by MAA-based biomaterials to effect
vascular regeneration.
What is unclear is how all of this from protein adsorption to neutrophil recruitment, to
macrophage polarization, to vascularization is determined by the properties of the biomaterial.
This thesis is a first step towards linking the pathways that are activated immediately following
protein adsorption (i.e., complement) to neutrophils, macrophage polarization and
vascularization. Follow up studies will need to link these components using more sophisticated
models. We attribute the vascular regenerating effect to the methacrylic acid and its strong
charge but how the properties of this charge (e.g., pKa) influence the adsorbed protein and
whether other similar anionic polymers would also promote an alternative host response and are
vascular regenerating is an as of yet, unanswered question.
55
Conclusion
This chapter demonstrated that inhibition of complement activation dampened some aspects of
the MAA-mediated host response; reduced neutrophil and macrophage infiltration was noted at
day 1 and at day 3, respectively. Furthermore, inhibition of complement activation eliminated the
MAA-mediated M2 macrophage polarization bias at day 3, but not at day 7. However, the
reduced MAA-mediated M2 macrophage polarization did not translate to reduced vessel density,
suggesting that complement activation and M2 macrophage polarization bias may not play vital
roles in MAA’s vascular regenerative mechanism (Fig. 14). However, more detailed studies are
needed to fully understand the role of complement and M2 macrophages in the context of MAA.
Finally, the subcutaneous model and techniques devised here may also prove to be a useful tool
in understanding the immediate host response to the implantation of various biomaterials.
56
Fig. 14. Effect of MAA beads on macrophage polarization, vascularization and the role of
complement activation. MAA beads promoted the formation of a denser and perfusable
vascular network when implanted subcutaneously, accompanied by an M2 macrophage
polarization bias, relative to controls. Inhibition of complement activation abated the M2
polarization bias at day 3, but not at day 7; no effect on MAA-mediated vascularization was
noted. While complement appeared to play some role in MAA-mediated vascularization, further
studies need to be conducted to fully understand its role.
57
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Appendices
S1. Gating Strategy for macrophages (day 3 shown; MAA beads). Single cells and live cells
were gated from all the cells. Leukocytes (CD45+) and non-leukocytes (CD45-) cells were gated
from the live, single cell population. From the CD45- non-leukocytes, endothelial (CD31+) cells
were gated. From the CD45+ gate, neutrophils (Ly6G+) were removed. Then, from the Ly6G-
gate, dendritic cells (CD11c+) were removed. Macrophages were gated on the Ly6G-CD11c-
population as F4/80+ and CD11b+. From the macrophage population, MHCII and CD206 were
used to distinguish between “M1” and “M2” macrophages, respectively.
65
Analysis Stain or antigen Type or fluorophore
Source (Clone/product code) Function
Histology CD31 Rabbit
polyclonal
Santa Cruz Biotechnology
(SC-1506)
Endothelial cell marker of
intercellular junctions.
F4/80 Rat
monoclonal
AbD Serotec (MCA497GA
Cl: A3-1)
Surface marker specific to
macrophages; involved in
peripheral T cell tolerance
Flow Cytometry Live/Dead Blue UV450 Invitrogen Discerns live cells from
dead cells.
CD45 (Leukocytes) BV711 BD Biosciences (30-F11) Especially expressed in
hematopoietic cells;
phosphatase involved in
cell growth and
differentiation.
CD11b (Macrophages) PE-eF594 BD Biosciences (M1/70) Regulator of leukocyte
adhesion, migration,
phagocytosis, and others.
F4/80 (Macrophages) APC-e710 eBioscience (BM8) See above.
Ly6G (Neutrophils) V450 BD Biosciences (1A8) A maturation marker
specific to neutrophils.
CD11c (Dendritic cells) BV511 BD Biosciences (HL3) An integrin associated
with adherence and
phagocytosis.
CD86 BUV395 BD Biosciences (B7-2) Provides “signal 2” for T
cell activation and
survival
CD206 BV650 or PE Biolegend (C068C2)
Biolegend (C068C2)
A mannose scavenger
receptor.
MHCII PerCP-e780 eBioscience (M5/114.15.2) Involved in antigen
presentation.
CD31 (Endothelial Cells) PE-Cy7 eBioscience (390) See above.
Unused, but mentioned
Arg1 arginase 1 N/A Enzyme upregulated in
M2 macrophages that
converts arginine to
ornithine
IFNγ interferon
gamma
N/A Potent inducer of M1
macrophage activation
IL-4 interleukin-4 N/A Primary inducer of M2
macrophage activation.
IL-10 interleukin-10 N/A An anti-inflammatory
cytokine that promotes
M2 macrophage activation
IL-13 interleukin-13 N/A An anti-inflammatory
cytokine that promotes
M2 macrophage
activation; shares same
receptor as IL-4.
S2. Markers used for immunohistochemistry and flow cytometry analyses and definitions.
List of markers used for analyses in addition to proteins and markers mentioned throughout the
document.
66
S3. Explant mass, cell number and normalized cell number for flow cytometry analyses.
(A) Mass of explants. (B) Numbers of cells collected by flow cytometry. (C) Normalized cell
numbers, obtained by dividing the cell number by the explant mass.
67
S4. Leukocytes, endothelial and dendritic cell populations in explanted tissues. Frequency of
CD45+ leukocytes (A) and CD45- non-leukocytes (B) expressed as a percentage of live cells.
(C) Normalized cell number and frequency of CD31+ endothelial cells, the latter expressed as a
percentage of CD45- leukocytes. (D) CD11c+ dendritic cells, expressed as a number (per mg of
explant) or as a percentage of CD45+ leukocytes.
68
S5. Expression of CD206, CD86, and MHCII in bone marrow-derived macrophages
polarized by IFNγ and IL-4. (A) Cells recovered from bone marrow cultured in M-CSF were
primarily macrophages. Black box designates F4/80+CD11b+ cells, gated on fluorescence-
minus-one (FMO) controls. (B) BMDM treated with IFNγ or IL-4. Note the shift in macrophage
expression of MHCII and CD206. Gating based on FMO controls. (C) Histograms depicting
CD86+, MHCII+, and CD206+ cells in BMDM polarized with IFNγ or IL-4. The numbers
represent the cells positive for the marker of interest, presented as a fraction of the parent
population (live cells (A) or macrophages (C)).
69
S6. Macrophage polarization - single positive cells. (A) Frequency of macrophages expressed
as a frequency of CD45+ leukocytes. (B-D) Normalized number and frequency (as a percentage
of macrophages) of (B) CD86+ cells, (C) MHCII+ cells and (D) CD206+ cells.
70
S7. Formation of giant-like cells in vitro. (A) Macrophages cultured on flat, non-tissue culture-
treated polystyrene and polarized with IFNγ or IL-4. (B) Expression of CD206 and MHCII in
macrophages recovered from flat, non-tissue culture-treated polystyrene. As expected, M(IFNγ)
“M1” macrophages expressed MHCII but not CD206 and M(IL-4) “M2” macrophages expressed
CD206 but not MHCII. (C) Macrophages cultured on tissue culture-treated polyethylene
terephthalate (PET) inserts over 72h in the presence of IL-4. Note the formation of larger, giant-
like cells that expressed MHCII+. The arrows indicate examples of the giant-like cells. (D)
Expression of CD206+ and MHCII+ in macrophages recovered from PET cell inserts. Note that
with IL-4 treatment, macrophages were primarily MHCII+ or MHC+CD206+, instead of the
conventional MHCII-CD206+. Scale bars = 100 μm. Giant cell fluorescence image courtesy of M.
Saleh.
S8. Gating strategy for validating dextran uptake in CD206+ macrophages. (A) Doublets
were removed, followed by dead cells. Two gating strategies were used to look at dextran+
macrophages: (B) Macrophages were identified first, then dextran+ macrophages, and lastly
CD206+ and MHCII+ dextran+ macrophages. (C) Dextran+ cells were identified first, then
macrophages, and lastly CD206+ and MHCII+ dextran+ macrophages. Note that not all
macrophages were associated with the dextran, but the majority of macrophages that were
associated with the dextran were CD206+, as identified in (B) and (C).
72
S9. Bone marrow harvest, macrophage culture and treatment with MAA beads or films. An in vitro
assay designed to investigate the effect of MAA on BMDM. (A) Bone marrow cells were harvested from
murine bone marrow, differentiated with M-CSF, and polarized with IFNγ or IL-4 on 10 cm petri dishes.
These cells were processed for flow cytometry to validate macrophage polarization (See Appendix, S5).
Additionally, non-polarized (M0) BMDM were re-plated onto 48 well plates, where they were treated
with MAA or MM beads, in the presence or absence of whole blood (0.5% v/v – 5 % v/v). These cells
were washed, fixed directly on the plate, and stained with DAPI, PE conjugated CD206 or PerCPe710
conjugated MHCII. The fluorescence signal was determined using a fluorescent plate reader. (B)
Schematic depicting the treatment of BMDM with MAA or MM films, instead of beads.
73
S10. Gating strategy for macrophages following inhibition of complement activation (day 7
shown; MAA beads). Single cells and live cells were gated from all the cells. Leukocytes
(CD45+) and non-leukocytes (CD45-) cells were gated from the live, single cell population.
From the CD45+ gate, neutrophils (Ly6G+) were removed. Then, from the Ly6G- gate,
macrophages were gated as F4/80+ and CD11b+. From the macrophage population, MHCII and
CD206 were used to distinguish between “M1” and “M2” macrophages, respectively.
74
S11. MAA beads modulated CD206, but not MHCII expression in the presence of blood.
(A) Macrophages cultured in IFNγ or IL-4 in 48 well plates and processed for fluorescence
scanning. As expected, M(IL-4) macrophages highly expressed CD206 but not MHCII while
M(IFNγ) highly expressed MHCII but not CD206. (B) BMDM treated with MAA or MM beads
for 24h in the presence or absence of 1% v/v whole mouse blood. Representative dot plots of
macrophages treated with MAA beads (C) or MM beads (D) and 0.5% whole blood (v/v) for
24h. Expression of CD206 (E) and MHCII (F) of macrophages treated with MAA or MM beads
in the presence of 0.5% v/v whole mouse blood. Treatment with MAA beads led to a small
increase in the expression of CD206, but not MHCII in BMDM. Conversely, treatment with MM
beads led to a small increase in the expression of MHCII, but not CD206. Uns = unstained.
75
S12. MAA films stimulated M2 marker Arg1 in M0 and M(IFNγ) cells. BMDM were
polarized, then treated with MAA or MM films for 72h. RNA from the cells were extracted,
reverse transcribed, then quantified. mRNA expression is expressed relative to NT controls and
ribosomal protein S18. (A) Arg-1 mRNA expression in BMDM polarized with IFNγ or IL-4 and
treated with MAA or MM films. (B) iNOS mRNA expression level in BMDM polarized with
IFNγ or IL-4 and treated with MAA or MM films. Treatment with MAA films increased the
expression of Arg-1, an M2 marker in M0 and M(IFNγ) cells. NT= no treatment. PCR
experiments conducted by I. Talior-Volodarsky.
76
S13. Administration of 4 mg/kg pentamidine or 2.5- 10 mg/kg ATA did not inhibit
complement activation over time. Serum from complement-inhibited mice were compared to
control mice administered 0.85% saline or normal chow (Ctrl). Complement-mediated red blood
cell hemolysis for animals administered 4 mg/kg pentamidine IP (A) or 2.5 – 10 mg/kg ATA SC
(B). (C) Efficiency of complement inhibition in ATA-treated animals, presented as the IC50. The
IC50 increased rapidly over time, indicating that the complement-inhibition effect of ATA was
short lived.
77
S14. Explant mass, cell number and normalized cell number in complement-inhibited
animals. (A) Mass of explants. (B) Numbers of cells collected by flow cytometry. (C)
Normalized cell numbers, obtained by dividing the cell number by the explant mass.
78
S15. Leukocytes and macrophage populations in complement-inhibited animals. (A-B)
Frequency (as a percentage of live cells) of CD45+ cells (A) and CD45-cells (B). (C) Frequency
(as a percentage of CD45+ cells) of Ly6G+ neutrophils. (D-F) Frequency (as a percentage of
F4/80+ macrophages) of CD206+ cells (D), CD86+ cells (E), and MHCII+ cells (F).
79
S16. Published manuscript: Lisovsky A, Zhang, DKY, Sefton MV, Biomaterials 2016. Reprinted
from [15] with permission from Elsevier.
lable at ScienceDirect
Biomaterials 98 (2016) 203e214
Contents lists avai
Biomaterials
journal homepage: www.elsevier .com/locate/biomater ia ls
Effect of methacrylic acid beads on the sonic hedgehog signalingpathway and macrophage polarization in a subcutaneous injectionmouse model
Alexandra Lisovsky a, 1, David K.Y. Zhang a, 1, Michael V. Sefton a, b, *
a Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Suite 407, Toronto, Ontario, Canada M5S 3G9b Department of Chemical Engineering and Applied Chemistry, University of Toronto, 164 College Street, Suite 407, Toronto, Ontario, Canada M5S 3G9
a r t i c l e i n f o
Article history:Received 12 February 2016Received in revised form14 April 2016Accepted 20 April 2016Available online 4 May 2016
Keywords:Methacrylic acidSonic hedgehogMacrophage polarization
* Corresponding author. Institute of BiomaterialsUniversity of Toronto, 164 College Street, Suite 407, T3G9.
E-mail address: [email protected] (M.V.1 These authors contributed equally.
http://dx.doi.org/10.1016/j.biomaterials.2016.04.0330142-9612/© 2016 Elsevier Ltd. All rights reserved.
a b s t r a c t
Poly(methacrylic acid-co-methyl methacrylate) (MAA) beads promote a vascular regenerative responsewhen used in diabetic wound healing. Previous studies reported that MAA beads modulated theexpression of sonic hedgehog (Shh) and inflammation related genes in diabetic wounds. The aim of thiswork was to follow up on these observations in a subcutaneous injection model to study the hostresponse in the absence of the confounding factors of diabetic wound healing. In this model, MAA beadsimproved vascularization in healthy mice of both sexes compared to control poly(methyl methacrylate)(MM) beads, with a stronger effect seen in males than females. MAA-induced vessels were perfusable, asevidenced from the CLARITY-processed images. In Shh-Cre-eGFP/Ptch1-LacZ non-diabetic transgenicmice, the increased vessel formation was accompanied by a higher density of cells expressing GFP (Shh)and b-Gal (patched 1, Ptch1) suggesting MAA enhanced the activation of the Shh pathway. Ptch1 is theShh receptor and a target of the pathway. MAA beads also modulated the inflammatory cell infiltrate inCD1 mice: more neutrophils and more macrophages were noted with MAA relative to MM beads at days1 and 7, respectively. In addition, MAA beads biased macrophages towards a MHCII�CD206þ (“M2”)polarization state. This study suggests that the Shh pathway and an altered inflammatory response aretwo elements of the complex mechanism whereby MAA-based biomaterials effect vascular regeneration.
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Methacrylic acid (MAA)-based biomaterials have a vascularregenerative effect in the absence of exogenous cells or growthfactors [reviewed in Refs. [1,2]]. These biomaterials were previouslyshown to promote vascularization [3e6], and improve myocuta-neous graft survival [4] and diabetic wound healing [5]. Togetherwith past work [6e9], the present study was aimed at further un-derstanding the mechanism behind this effect. We presume thatsuch biomaterials drive an “alternative foreign body response” thatis distinct from the fibrosis associated with the classical foreignbody response.
Gene expression analysis of diabetic wounds treated with
and Biomedical Engineering,oronto, Ontario, Canada M5S
Sefton).
poly(methacrylic acid-co-methyl methacrylate) (MAA) beadsshowed an over fourfold upregulation in the expression of the sonichedgehog (Shh) gene [6], which has been implicated in adultvascularization [10,11]. The Shh pathway is also activated duringinflammation [12] and has been shown to polarize macrophagestowards an alternatively-activated, wound healing (“M2”) pheno-type [13]. Although, MAA-based biomaterials did not modulate theexpression of classical angiogenic genes (e.g., vascular endothelialgrowth factor (VEGF)) [6e8], MAA beads modulated inflammation-associated genes (e.g., interleukin 1b (IL1b)) in diabetic wounds [6],in an air pouch model [9] and macrophage-like cells (dTHP1 cells)in vitro [7,8]. A phosphoproteomics study of dTHP1 cells treated fora few minutes with a MAA-based biomaterial distinguished anumber of phosphorylated proteins involved in macrophage po-larization (e.g., solute transporter monocarboxylate transporter 4)among the many phosphorylated proteins that were differentiallyregulated between a MAA-based and a control biomaterial [14].Overall, these results led to the hypothesis that MAA-based bio-materials modulate the Shh signaling pathway and inflammatory
A. Lisovsky et al. / Biomaterials 98 (2016) 203e214204
cell responses, including macrophage polarization. Hence, a sub-cutaneous injection model was devised to investigate the effects ofMAA beads on the host response without the confounding factorsof diabetic wound healing. To investigate the activation of the Shhpathway specifically, MAA beads and control poly(methyl meth-acrylate) (MM) beads were injected subcutaneously in transgenicShh-Cre-eGFP/Ptch1-LacZ mice. In these mice the expression of thereporters, GFP and b-Gal, was shown to be consistent with thepattern of Shh and Ptch1 mRNA expression, respectively [15,16].The activation of the Shh signaling pathway was suggested as thedensities of both GFPþ (Shh) and b-Galþ (patched 1, Ptch1, thetarget of the pathway) cells were upregulated by treatment withMAA beads in this model. To investigate inflammatory cell re-sponses, bead implants were analyzed by immunohistochemistryand flow cytometry for the number and polarization state of infil-trating inflammatory cells. MAA beads increased the density ofneutrophils at day 1 and macrophages at day 7 and biased mac-rophages towards the MHCII�CD206þ state representative of the“M2” phenotype. We also compared the therapeutic effect of abiomaterial in both males and females illustrating a difference inresponse between sexes.
2. Methods
2.1. MAA and MM bead preparation
Poly(methacrylic acid-co-methyl methacrylate) (MAA-co-MMAor MAA) beads were composed of 45 mol% methacrylic acid(Sigma-Aldrich Canada Ltd., Oakville, ON, Canada), 1 mol% ethyleneglycol dimethacrylate (Sigma-Aldrich Canada Ltd.) and 64 mol%methyl methacrylate (Sigma-Aldrich Canada Ltd.). MAA beads weresynthesized by suspension polymerization as previously described[4] and were sieved to obtain beads in the diameter range of150e250 mm. Methacrylic acid content of the synthesized beadswas confirmed by titration. Control poly(methyl methacrylate)(MM) beads (same diameter) were obtained from Polysciences(Warrington, PA). Beads were washed in either 95% ethanol (MAAbeads) or 1 N HCl (MM beads) repeatedly and then rinsed five timesin LAL reagentwater (MJS Biolynx Inc., Brockville, ON, Canada) priorto use in vivo. Analysis with a limulus amebocyte lysate (LAL)pyrochrome endotoxin test kit (Cape Cod Inc., Falmouth, MA)indicated that beads contained <0.25 EU/100mg. Elemental surfacecomposition analysis (ThermoFisher XPS, Surface-InterfaceOntario, University of Toronto) showed minimal Si contamination(~0.07%) and that measured surface composition (atom%) was closeto the theoretical expectation. MAA beads had a rough, poroussurface, were negatively charged and did not degrade over timein vivo; MM beads were smooth [4,5].
For subcutaneous injections, a 1 mL syringe with an 18 gaugeneedlewas loadedwith either 5mgMAA beads or 15mgMMbeads(or no beads, vehicle control) suspended in 250 mL of 50% w/vpolyethylene glycol (PEG, avg. mol. wt. 1450, sterile-filtered; Sigma-Aldrich Canada Ltd.) in PBS. The 1:3 wt ratio (5 mg MAA:15 mg MM) was used to account for MAA bead swelling upon hy-dration at physiological pH [4] to approximately equate implantedvolumes. Vehicle control was used only for the flow cytometrystudy because the vehicle control implant area could not be definedreproducibly for vessel and cell density analyses.
2.2. Animals
All animal work was done with the approval of the University ofToronto Animal Care Committee. Animals were housed understerile conditions in the University of Toronto's Department ofComparative Medicine. The experiments were done with CD1 mice
(6e8 week old, males, Charles River Laboratories, MA) and Shh-Cre-eGFP/Ptch1-LacZ mice (10e12 week old, males and females).Shh-Cre-eGFP/Ptch1-LacZ heterozygous mice of CD1 backgroundwere bred in house by crossing CD1 females (Charles River Labo-ratories, MA or bred in house) with Shh-Cre-eGFP/Ptch1-LacZheterozygous males. The original Shh-Cre-eGFP/Ptch1-LacZ malewas donated by Professor Chi-chung Hui (Hospital for Sick Chil-dren, Toronto, ON, Canada) and created by crossing Shh-Cre-eGFP[15] with Ptch1-LacZ [16] mice. Shh-Cre-eGFP mice were createdby inserting a gfpcre cassette at the ATG of Shh; expression of GFPprotein was reported to colocalize with Shh mRNA [15]. Ptch1-LacZmice were developed by inserting the LacZ gene into Ptch1; theexpression of the reporter was consistent with the pattern of Ptch1transcription [16].
Mice were genotyped to detect the presence of Shh-Cre-eGFPand Ptch1-LacZ mutations. DNA from ear notches was extractedby alkaline lysis. Primers (Supplementary Information, Table 1)were synthesized by Sigma Genosys (Sigma-Aldrich Canada Ltd.)and prepared by resuspension in RNase/DNase free water. PCR re-actions are detailed in the Supplementary Methods [80].
2.3. Subcutaneous injection
Mice were anesthetized with 0.5% w/v isofluorane prior tosurgery and an analgesic (Ketoprofen, 5 mg/kg) was administeredintraoperatively. The dorsal area of a mouse was shaved and theremaining hair was removed either by waxing (Nair wax strips) orby hair removal cream (Veet). The skin was sterilized with 70%ethanol and Betadine. An 18-gauge needle was used to inject MAA,control MM beads or vehicle (PEG). Two injections on either side ofthe dorsumwere performed for each mouse. A small subcutaneouspocket was made with the needle on the side of the dorsum bymoving the syringe from side to side, while deliberately attemptingto nick small blood vessels to promote injury prior to injection(Fig.1). Following surgery, mice were housed individually, fed chowand water ad libitum, and monitored for any signs of discomfort. At1e7 days post-injection, the mice were sacrificed using CO2, fol-lowed by cervical dislocation. The implants were removed surgi-cally and processed for histology, imaging or flow cytometry.
2.4. Histology and immunohistochemistry
Immediately upon euthanizing mice, the bead implant andseveral mm of surrounding tissue was excised from the right side ofthe dorsum and fixed in formalin. Tissue samples were embeddedin deep paraffin blocks, cut into sections, processed and stainedwith hematoxylin and eosin (H&E), Masson's trichrome, CD31, GFP,b-galactosidase (b-Gal), F4/80 and CD206 (SupplementaryInformation, Table 2). Histology slides were scanned (20�) usingan Aperio ScanScope XT (LeicaMicrosystems, Concord, ON, Canada)by the Advanced Optical Microscopy Facility (AOMF, Toronto, ON,Canada).
The scanned slides were analyzed using Aperio ImageScope(Version 11) at 4 and 7 days post-implantation. For vessel counts, aregion of interest (ROI) was defined by measuring a distance of500 mm around each cluster of beads. CD31þ vessel-like structures(criterion being the presence of a lumen)were counted in the tissuewithin this defined region. The vessel density was calculated bydividing the total number of vessels by area of the ROI. For othercounts (GFP, b-Gal, F4/80 and CD206), a distance of 200 mm aroundbead clusters was used to define the ROI. The cell density wascalculated by dividing the total number of positive cells by the areaof the ROI.
Fig. 1. Subcutaneous injection model for investigating cellular and molecular mechanisms of vascular regenerative MAA-based biomaterials. MAA beads, control MM beads orvehicle (PEG) were injected on both sides of the dorsum of mice. At 1e7 days post-injection, the tissues were removed and processed for histology, imaging and flow cytometry. Inthe future, the model will also be used for molecular analysis and inhibition studies. The image is a scanning electron micrograph of MAA beads.
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2.5. CLARITY preparation and imaging
Seven days following subcutaneous injection of MAA or MMbeads, Alexa 647-conjugated lectin (GSL-1: Griffonia (Bandeiraea)Simplicifolia; 100 mg in 150 mL PBS; Vector Laboratories, Burlington,ON, Canada) was injected via tail vein 10 min prior to sacrifice.Fluorophore conjugation was performed in-house using Alexa 647modified with a NHS-ester chemistry [17]. GSL-1 is a mouse-specific lectin which binds to the galactosyl residues of mouseendothelial cells, enabling labeling and visualization of the mousevasculature [18]. The implants with the surrounding subcutaneoustissue were removed surgically and processed using a modifiedCLARITYprotocol for confocal imaging [19]. Explants were fixed in asolution containing 4% acrylamide (Sigma-Aldrich Canada Ltd.), 4%paraformaldehyde, 0.05% bis-acrylamide (Sigma-Aldrich CanadaLtd.) and 0.25% (w/v) VA-044 thermal initiator (Sigma-AldrichCanada Ltd). After one week of incubation, the acrylamide waspolymerized at 37 �C for 3 h. Polyacrylamide-embedded explantswere cleared for 14 days at 50 �C in the clearing solution (8% SDS inborate buffer, pH 8.5; eBioscience, San Diego, CA), which waschanged every 2nd day. Post-clearing, the explants were counter-stained with SYTOX green nucleic acid stain (100 pmol/mg; LifeTechnologies, Burlington, ON, Canada) for 48 h. Refractive indexmatching was performed by infusing explants with 70e75% 2,20-thiodiethanol in borate (adjusted to pH 10; Sigma-Aldrich CanadaLtd.) [21]. Explants were imaged using a Nikon A1 confocal mi-croscope (Nikon, Melville, NY) at the Center for Microfluidics Sys-tems (University of Toronto).
2.6. Digestion of tissue explants
Subcutaneous tissue containing injected beads was separatedfrom the skin and muscle layers. For PEG samples, subcutaneoustissuewas explanted in the samemanner using the injection needlewound site as a guide. Tissues were weighed and then digestedfollowing a previously described digestion protocol [22]. Briefly,samples were finely minced in 500 mL of 1 X HBSS containing450 U/mL collagenase I (Sigma-Aldrich Canada Ltd.), 125 U/mLcollagenase XI (Sigma-Aldrich Canada Ltd.), 60 U/mL DNase I
(Sigma-Aldrich Canada Ltd.), 60 U/mL hyaluronidase (Sigma-Aldrich Canada Ltd.) and 1 M HEPES (Sigma-Aldrich Canada Ltd.).The sample was incubated in this solution for 1 h at 37 �C andhomogenized using a gentleMACS Octo Dissociator (Miltenyi BiotecInc., San Diego, CA). Tissue was further digested for 60 min at 37 �Cand 250 rpm. The cell suspension was filtered using a 40 mm cellstrainer (Fisher Scientific, Ottawa, ON, Canada) to remove beadsand debris. The remaining cells were washed in PBS supplementedwith 0.5% BSA and 2 mM EDTA, pelleted and stained with live/deadstain, CD11b, CD11c, CD206, CD31, CD45, CD86, F4/80, Ly6G, MHCII(Supplementary Information, Table 2). All antibodies were dilutedaccording to the manufacturers' recommendations and titrated inhouse to optimize staining.
2.7. Flow cytometry analysis of tissue explants
The gating strategy (Supplementary Information, Fig. 1) was asfollows: after isolating live single cells, CD45 distinguished leuko-cytes from non-leukocytes. Neutrophils were identified as Ly6Gþand dendritic cells as CD11cþLy6G�. Macrophages were firstidentified as CD11c�Ly6G�F4/80þCD11bþ and then further char-acterized as MHCIIþ CD206� (“M1”) and MHCII�CD206þ (“M2”).Endothelial cells were identified as CD31þCD45�. Cells were gatedaccording to positive staining for each antibody using fluorescenceminus one (FMO) controls. Cell populations were expressed eitheras a percentage or as a normalized value: estimated total number ofcells divided by the weight of the explanted tissue. The number ofcells was estimated from the flow cytometry results with 123counteBeads (eBioscience) used to determine cell recovery (~50%).
2.8. Statistical analysis
All data are presented as mean ± standard error of the mean(SEM). Statistical analysis was performed using IBM SPSS Statistics(Version 22). Levene's test was used to test for equality of varianceamong samples. Statistical comparison between two treatmentgroups (MAA, MM) was performed using independent t-test forsamples with equal variances (Levene's test: p > 0.05) and Mann-Whitney test for samples with significantly different variances
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(Levene's test: p < 0.05). Statistical comparison among threetreatment groups (MAA, MM, PEG) was performed using two-wayANOVA followed by Tukey's post hoc test for significance. The levelof statistical significance was set at p < 0.05.
3. Results
3.1. Subcutaneous injection model
Previously, the vascular regenerative MAA beads were investi-gated in vivo in wounds of diabetic db/db mice [5,6]. During woundhealing, MAA beads became entrapped in a scab, which at times felloff prior to the studied time points, preventing the identification ofthe cells that were associated with the beads. A subcutaneous in-jection model was developed to directly interrogate cellular re-sponses and vascularization, while simplifying the host response;injections were less invasive compared to full-thickness wounds.An additional advantage of this model was the ability to studyMAA-mediated changes in the cellular infiltrate. Consistent withthe previous studies with MAA-based biomaterials that had onlyused males, flow cytometry studies were also done only in males.However, since females have been shown to respond differently tovarious diseases and treatments [reviewed in Refs. [23,24]], histo-logical analyses were done in bothmale and female transgenicmiceand sex differences were noted.
The earlier study showed that Shh gene expression was upre-gulated more than fourfold compared to the controls (MM beadsand no treatment) in diabetic wounds [6], but this did not showthat the pathway was indeed engaged. Motivated by this result, weinvestigated the expression of the Shh pathway in non-diabeticShh-eGFP-Cre/Ptch1-LacZ CD1 mice. In these double heterozy-gous mice, the expression of reporters (GFP and b-Gal) was shownto be consistent with the patterns of Shh and Ptch1 mRNA expres-sion [15,16], thus allowing the investigation of cells expressing GFP(Shh) and b-Gal (Ptch1) in the tissue surrounding the beads. Weused the corresponding CD1 mice to investigate the effect of MAAbeads on inflammatory cells, with a focus on macrophages.
3.2. The effect of MAA beads on vessel density in males and females
As expected, MAA beads promoted vascularization in the tissuesurrounding the beads in non-diabetic mice (Fig. 2A,Supplementary Information, Fig. 2). MAA beads increased CD31þvessel formation at day 7 in both males (p ¼ 0.013, Fig. 2B) andfemales (p ¼ 0.009, Fig. 2C), although the effect in females was notas pronounced as in males. At day 4, MAA beads appeared to in-crease the CD31þ vessel density in males, but this difference(relative toMM) was not statistically significant (p¼ 0.085, Fig. 2B).Compared to females, males had an increased level of vascularity atday 4 (p ¼ 0.024) and similar vessel densities at day 7 in MAA-treated mice. No differences were noted between sexes in theirvascular response to control MM beads.
CD1 male mice were injected (via the tail vein) at day 7 with amouse Alexa 647-conjugated lectin (GSL1, a lectin that labels themouse endothelium) to visualize perfused blood vessels in the vi-cinity of the beads (Fig. 2D). To increase the depth of imaging, ex-plants were processed using a CLARITY protocol [20]. To ourknowledge, this is the first use of the technique to investigate host-biomaterial interactions. Explanted tissues fromMAA-treated miceshowed high levels of Alexa 647-GSL1 staining surrounding MAAbeads, consistent with the greater levels of vascularizationobserved with the histological analysis. In explanted tissues fromMM-treated mice, lectin staining was not seen around the beadsand was primarily seen in the skin far from the beads. A thick layerof cells surrounded MM (Fig. 2D) but not MAA beads, suggested an
alternative cellular response. Similarly, a dense layer of cells wasseen in Masson's trichrome images (Fig. 2A). Taken together, theseresults suggested that MAA beads were effective in promotingvascularization in both sexes (in non-diabetic mice) and that theMAA-induced vessels were perfusable.
3.3. The effect of MAA beads on the Shh signaling pathway
After the subcutaneous injection of beads in transgenic mice,GFPþ and b-Galþ cells were found in close proximity to theinjected beads (Fig. 3A and B, Supplementary Information, Fig. 3).At day 4, MAA beads increased the expression of GFPþ (p ¼ 0.001)(Fig. 3C) but not b-Galþ cells relative to control MM beads (Fig. 3D).At this time, both males and females showed similar numbers ofGFPþ and b-Galþ cells, whether animals were treated with MAA orMMbeads; thus, for day 4 the data for both sexes were combined toachieve a higher n. At day 7, the MAA-induced upregulation of GFPwas seen in females (and not males); the increase (p ¼ 0.051) justmissed our criterion for statistical significance (Fig. 3C). MAA-treated mice showed greater b-Gal protein expression at day 7 inmales (p ¼ 0.026) but not females (Fig. 3D). There was a significantincrease in b-Gal (p < 0.001) from day 4 to 7 and no change wasnoted for GFP over time (Fig. 3C and D). Together, these data sug-gested thatMAA beads enhanced the activation of the Shh signalingpathway, as Ptch1 is the target of the Shh signaling pathway[reviewed in Ref. [25]].
Some of the GFPþ and b-Galþ cells exhibited a rounded shape(Fig. 3A and B), suggesting that macrophages may have beeninvolved in the Shh pathway modulation with MAA beads.Consistent with this observation, in vitro mouse bone marrow-derived macrophages treated with MAA-based films increasedthe expression of the Shh gene more than fourfold compared tomacrophages left untreated (Supplementary Information, Fig. 4,p ¼ 0.035); control MM films did not upregulate Shh.
3.4. The effect of MAA beads on inflammatory cell recruitment
To further investigate the effect of MAA beads on macrophages,the pan macrophage marker F4/80 and M2 marker CD206 wereused to quantify macrophages surrounding beads after subcu-taneous injection (Fig. 4A and B). A dense ring of F4/80þ cells wasseen surrounding MM beads (Fig. 4A), similar to that present in theMasson's trichrome and CLARITY-processed images (Fig. 2A and D);this dense ring was rarely seenwith MAA beads. Nonetheless, therewas no difference in F4/80þ cell density between MAA and MMbeads at either time point (Fig. 4C) consistent with a differentdistribution of cells; no differences were seen in comparing malesand females. CD206þ cells were closely associated with both beadtypes (Fig. 4B). Treatment with MAA beads resulted in a higher celldensity of CD206þ cells in females (and not males) at day 4(p ¼ 0.013) compared to MM beads (Fig. 4D). There was an increasein CD206þ cells in males at day 7 with MAA beads (p ¼ 0.066), butthe increase was not statistically significant (Fig. 4D); there was nodifference at day 7 in females.
Flow cytometry was used to follow up on these observations. Anearlier time point (day 1) was added to investigate neutrophilrecruitment. There was no statistical difference in the mass of tis-sue explants (Supplementary Information, Fig. 5A), estimated totalnumbers of collected cells (Supplementary Information, Fig. 5B)and normalized cell numbers (Supplementary Information, Fig. 5C)among treatment groups (MM beads, MAA beads, and PEG vehicle)at all studied time points. Using the gating strategy illustrated inthe Supplementary Fig. 1, higher densities of CD45þ cells werenoted in both bead explants compared to the PEG vehicle control atday 1 (Fig. 5A; Supplementary Information, Fig. 6A) with a
Fig. 2. Vessel formation in mice injected with vascular regenerative MAA beads. (A) Histology sections of Shh-eGFP-Cre/Ptch1-LacZ CD1 mice treated with MAA or MM beads at day7 stained with CD31 (left) and Masson's trichrome (right). Arrows indicate examples of vessels. (B, C) Tissues treated with MAA beads in transgenic mice had a significantly highervessel density in males (B) and females (C) at day 7. (D) Confocal microscopy image of CLARITY-processed tissues treated with MAA and MM beads from non-transgenic CD1 micestained with Alexa 647-GSL1, a lectin specific for mouse vasculature, and Sytox Green. Perfused vessels weaved around MAA beads but not MM beads. Most of the vessels in MM-treated mice were found in the skin further away from the beads. Scale bars ¼ 200 mm. n ¼ 3 (day 4), n ¼ 6 (day 7); *p < 0.05; **p < 0.01. (For interpretation of the references tocolour in this figure legend, the reader is referred to the web version of this article.)
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corresponding higher number of CD45� (cells other than leuko-cytes) in vehicle explants (Fig. 5B; Supplementary Information,Fig. 6B). More CD45� cells were noted in tissues treated withMM compared to MAA beads at day 7 (Fig. 5B).
For the biomaterial treatment groups, neutrophils were mostprevalent at day 1 post-injection and as expected, their estimatednumbers declined dramatically at days 3 and 7 (Fig. 5C). MAA beadssignificantly increased neutrophil recruitment (p < 0.0001)compared to both controls at day 1 (Fig. 5C). More macrophageswere found in MAA-treated mice compared to both MM and PEGcontrols at day 7 (p < 0.05) (Fig. 5D). The estimated number ofmacrophages in theMM- and PEG-treatedmice decreased from day3 to day 7; this effect was not observed with MAA beads (Fig. 5D).The intensity of F4/80 expression by macrophages varied dramat-ically from day 1 to days 3 and 7 (Fig. 5E). Endothelial and dendriticcells were also quantified by flow cytometry. No statistically sig-nificant differences were noted in the density of endothelial cellsamong the treatment groups (Supplementary Information, Fig. 6C).At day 7, the frequency of dendritic cells increased in explants forboth bead types (Supplementary Information, Fig. 6D) relative tovehicle control. Overall, these results indicated a differential in-flammatory cell response to MAA beads compared to biomaterialand vehicle controls. Treatment with MAA beads increased therecruitment of neutrophils at day 1 and the number of
macrophages at day 7.
3.5. The effect of MAA beads on macrophage polarization
The flow cytometry protocol was used to distinguish macro-phage polarization states: “M1” using MHCII and CD86 as markersand “M2” using CD206 [reviewed in Refs. [26e28]] (SupplementaryInformation, Fig. 7). While the expression of CD86 was similaramong all three treatment groups (Supplementary Information,Fig. 7B), there were some significant differences in the numberand frequency of macrophages expressing MHCII (SupplementaryInformation, Fig. 7C) and CD206 (Supplementary Information,Fig. 7D); CD86 was excluded from further analyses.
The polarization bias was reflected in the representative dotplots for both biomaterials (Fig. 6A and B). Treatment with MAAbeads significantly increased MHCII-CD206þ (M2) macrophagesand decreased MHCIIþCD206� (M1) macrophages compared toboth MM and PEG controls at day 7 (p < 0.01) (Fig. 6C). Conversely,treatment with control MM beads had an opposite effect withsignificantly more M1 and fewer M2 macrophages relative to MAAbeads at day 7 (p < 0.01) (Fig. 6D). By day 7, the majority of mac-rophages in MM-treated mice expressed either both markers(MHCIIþCD206þ) or neither marker (MHCII�CD206�) (Fig. 6E andF). The frequency of cells expressing a double-positive phenotype
Fig. 3. Modulation of the GFPþ and b-Galþ cell density after subcutaneous injection of MAA beads in Shh-eGFP-Cre/Ptch1-LacZ CD1 mice. (A, B) Serial sections of day 4 tissuestreated with MAA beads, stained for GFP (A) or b-Gal (B). Arrows show examples of cells positive for the marker of interest. (C, D) Density of GFPþ (C) and b-Galþ (D) cells at days 4and 7. Treatment with MAA beads increased the density of GFP-expressing cells but not b-Gal-expressing cells at day 4; there was no difference between males and females at day 4.At day 7, the density of cells expressing b-Galþ was upregulated in males (but not females) and the density of cells expressing GFP was greater in MAA-treated females but thedifference was not significant (p ¼ 0.051). There were more b-Galþ cells at day 7 than at day 4 in both sexes and with both materials; there was no substantive effect of time on thenumber of GFPþ cells. Scale bars ¼ 200 mm. n ¼ 6; *p < 0.05; **p < 0.01.
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increased progressively from day 1 to day 7 in MM but not in MAA-treated mice (Fig. 6F). On the other hand, in the MAA-treated mice,the majority of macrophages were consistently MHCII�CD206þ(M2) from day 3 onwards to day 7 (Fig. 6E and F). Together, theseresults suggested that MAA beads biased macrophages towards aM2 polarization state.
4. Discussion
4.1. The effect of MAA beads on vascularization
Previous studies in diabetic male mice (BKS.Cg-mþ/þ Leprdb/Jmice, db/db) showed that MAA beads increased vascularization incutaneous wounds [5,6]. In agreement with these studies, subcu-taneous injection of MAA beads increased vessel density in non-diabetic mice compared to control MM beads (Fig. 2), high-lighting the vascular potency of MAA beads even in the absence ofthe physiological need present during diabetic wound healing.MAA beads nearly doubled the number of vessels at day 7 (~90%increase) (Fig. 2B) in males. Other synthetic biomaterials have alsobeen shown to improve vascularization without the addition ofexogenous factors [29e31]. For example, changing porosity(decrease from 60 mm to 30 mm) resulted in ~40% increase in vesselformation with poly(2-hydroxyethyl methacrylate, poly(HEMA))scaffolds implanted into the myocardium of male rats [29].
In females, the effect of MAAwas more modest with an increaseof only about 30% at the same time point (Fig. 2C). The strongerMAA-mediated vascular response seen in males was most likelydue to sex-based hormone differences [32,33]. The Shh pathway isregulated by estrogen [34,35] and hence differences in the extent ofShh pathway activation between males and females may accountfor the higher vascularization induced by MAA beads. Males had ahigher density of b-Galþ (Ptch1) cells than females at day 7(Fig. 3D). The inclusion of females in this study increased ourappreciation for the effect of sex on the host response to MAA-based biomaterials, consistent with efforts to decrease sex biasand improving the quality and reproducibility of preclinical
research [reviewed in Refs. [36,37]], here in the context of thera-peutic biomaterials design.
To investigate the perfusability of newly formed vesselsfollowing the MAA bead treatment, the bead explants were pre-pared using the CLARITY protocol (Fig. 2D). During CLARITY pro-cessing, fatty lipids were removed while proteinaceous structuresand morphology were retained, enabling deep imaging and 3Dvisualization of these fragile tissues [20,38]. Alexa 647-GSL1 (viatail vein injection) staining was only observed aroundMAA and notMM beads (Fig. 2D) indicating that the MAA-induced vessels wereperfusable.
4.2. The effect of MAA beads on Shh signaling
During Shh pathway activation, binding of Shh to its receptorPtch1 relieves the inhibitory effect of Ptch1 on Smoothenedresulting in the activation of the Gli transcription factors respon-sible for downstream modulation of Shh target genes [reviewed inRef. [25]]. Previously, MAA beads were shown to upregulate Shh(and perhaps Gli3) gene expression in diabetic wounds at day 4(although the latter was not significant, p ¼ 0.052) [6]; Gli3 is atranscription factor associated with the angiogenic effect of the Shhpathway [39]. Because the diabetic wound healing milieu wascomplex, it was challenging to investigate the differential expres-sion of other genes associated with the Shh pathway in thesewounds. The transgenic Shh-Cre-eGFP/Ptch1-LacZ mouse modelwas used to enable quantification of cells expressing GFP or b-Galproteins (Fig. 3A and B), rather than just gene expression within awound as a whole. This study suggested, for the first time, that theShh pathway was indeed modulated by MAA beads: MAA-treatedmice had a significant increase in the expression of both GFP(Shh) and b-Gal (the target of Shh signaling) (Fig. 3C and D). Theactivation of the Shh signaling pathway is presumed to be oneaspect of the mechanism of the vascular regenerative effect ofMAA-based biomaterials.
Shh is well known for its role in embryogenesis, carcinogenesis[reviewed in Refs. [25,40]] and more recently for its involvement in
Fig. 4. Macrophage infiltration after treatment with MAA beads, by histology. (A) Tissue section from MAA- and MM-treated mice stained with pan macrophage F4/80 marker atday 4. A dense ring of F4/80þ cells (macrophages) surrounded control MM beads but not MAA beads. (B) Serial sections stained with F4/80 and CD206 (M2 macrophage marker)fromMAA-treated mice at day 4. CD206þ cells were closely associated with the beads. Arrows show examples of cells positive for the marker of interest. (C, D) Density of F4/80þ (C)and CD206þ (D) cells in tissues following treatment with MAA and MM beads at days 4 and 7 in males and females. CD206þ cell density was increased in females at day 4. Therewas no substantive effect of time on the number of F4/80þ and CD206þ cells in both sexes in MAA-treated mice. Scale bars ¼ 200 mm. n ¼ 6 (except for CD206 at day 4, n ¼ 3);*p < 0.05.
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Fig. 5. Analysis of inflammatory cells in tissues treated with MAA beads, MM beads or PEG vehicle (CD1 mice). (AeD) Estimated number of CD45þ leukocytes (A), CD45� cells otherthan leukocytes (B), Ly6GþCD11bþCD45þ neutrophils (C) and F4/80þ CD11c-Ly6G-CD11bþCD45þ macrophages (D). MAA beads significantly increased the number of CD45þ cells(A) and neutrophils (C) at day 1 and macrophages at day 7 (D), while decreasing the number of CD45� cells at day 7 (B). (E) F4/80 and CD11b expression in CD45þ cells at day 1 andday 7; note the F4/80 mean fluorescent intensity increased over time. The F4/80 and CD11b gate (black box) was set based on fluorescence minus one (FMO) negative controls. n ¼ 4(except MM and PEG for day 1, n ¼ 3); *p < 0.05, ***p < 0.001.
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adult vascularization, inflammation andwound healing [10,41e43].Intramuscular delivery of Shh improved blood flow in a limb[10,44] while inhibition of endogenous Shh abolished angiogenesis[45] in mouse models of hind limb ischemia. The Shh pathway wasalso shown to be activated in skeletal muscle post-injury and itsinhibition resulted in decreased vascularity and increased fibrosisand inflammation [46]. During ischemic injuries, onemechanism ofShh-induced vascularization is via upregulation of angiogenic andvasculogenic factors (e.g., VEGF, angiopoietins) [44,46e48]. Inter-estingly, our previous studies indicated that MAA-based bio-materials did not alter the expression of classical angiogenic genes(e.g., VEGF) in diabetic wounds in vivo [6] and in macrophages(dTHP1) [7,8] and endothelial cells (HUVEC) [7] in vitro. At the sametime, MAA-based biomaterials upregulated inflammation-associated genes [6e9] and earlier in vivo work in diabeticwounds [6] and an air pouch model [9] suggested that MAA beadsaltered the foreign body response.
As such, the MAA-mediated Shh vascular effect may haveinvolved inflammation [11,12]. Qualitative observations of GFPþ(Shh) and b-Galþ (Ptch1) cells indicated that some of the cellspositive for these markers had round macrophage-like cellmorphology (Fig. 3A and B). Additionally, in vitro treatment of bonemarrow derived macrophages with MAA-based films upregulatedthe Shh gene fourfold compared to no treatment (no film) control(Supplementary Information, Fig. 4). The Shh signaling pathwayhas been shown to be activated in inflammatory cells in response toinflammation during injury [43] and host-pathogen interactions[12,13]. LPS upregulated Shh expression in brain astrocytes [43] anda monocyte cell line (THP-1) [49], while infection with a pathogeninduced upregulation of Shh, Ptch1 and Gli transcription factors inmacrophages [12].
This study demonstrated that MAA beads increased the numberof inflammatory cells (Fig. 5A) at day 1 compared to biomaterial andvehicle controls indicative of a stronger initial inflammatory
response. In addition, previous work showed that MAA beads [8]and films [7] upregulated two important mediators of inflamma-tion e tissue necrosis factor a (TNFa) [reviewed in Ref. [50]] andinterleukin 1b (IL1b) [reviewed in Ref. [51]], both of which havebeen implicated in modulation of Shh signaling [12,43,52,53]. Shhpathway activation was decreased in TNFa-null macrophages [12]and increased with exogenous TNFa treatment [12,53]. In amouse model with depleted CD11bþ macrophages, the Shhresponse was reduced but partially rescued by the injection of IL1bat the time of injury [43]. Thus, Shh signaling may have beenupregulated by MAA-mediated inflammatory signals. At the sametime, once activated, the Shh pathway has also been implicated inmodulating inflammation [12,54,55], specifically macrophage po-larization towards the M2 state [13] (discussed in Section 4.4).
4.3. The effect of MAA beads on inflammatory cell infiltration
MAA beads altered the inflammatory cell landscape relative tocontrols (Fig. 5). As expected, the presence of a biomaterial (eitherMAA or MM beads) resulted in more CD45þ leukocytes relative tothe PEG vehicle control (Supplementary Information, Fig. 6A). Afourfold increase in Ly6Gþ neutrophils was evident at day 1 inmiceinjected with MAA beads relative to both controls (Fig. 5C). The linkbetween MAA beads and neutrophil infiltration is not well under-stood but may have been a result of protein (e.g., complement)adsorption differences [56]. The significance of the increase inCD11cþ dendritic cells (Supplementary Information, Fig. 6D) is notclear. One caveat with the data is that the total number of cells wasdetermined by flow cytometry, with calibration beads used todetermine the ratio between number of events and number of cells.Cell numbers were further normalized by the mass of the explants,recognizing that the volume of tissue that was digested varied fromsample to sample. The reported numbers are reasonable estimatesof cell numbers, recognizing that we are interested in differences in
Fig. 6. Analysis of polarization states in recovered explants (CD1 mice). (A, B) Representative dot plots of F4/80þ cells (macrophages) at day 3 in mice treated with MAA (A) and MMbeads (B). (C, D) The number and frequency of the individual single positive, double positive, and double negative MHCII or CD206 macrophage populations in mice treated withMAA beads, MM beads or PEG vehicle control. (C) Normalized number and frequency of MHCII-CD206þ (“M2”) macrophages. (D) Normalized number and frequency ofMHCIIþ CD206- (“M1”) macrophages. MAA beads biased macrophages towards a M2 polarization state; noted by a progressive increase in M2 macrophages, relative to MM beads.(E, F) Distribution of polarized macrophages: normalized number (E) and frequency (F) of macrophages that were MHCII-CD206þ, MHCIIþCD206þ, MHCIIþCD206�, andMHCII�CD206�. n ¼ 4 (except MM and PEG for day 1, n ¼ 3); *p < 0.05, **p < 0.01, ***p < 0.001.
A. Lisovsky et al. / Biomaterials 98 (2016) 203e214 211
inflammatory cell infiltration over the course of the study. Thenormalization protocol may account for the apparent increase inCD45� cells seen with PEG at day 1 (Fig. 5B). Explant masses andtotal cell numbers were low (Supplementary Information Fig. 5)with the vehicle-only controls so that after normalization, thenormalized numbers were artificially high. Following PEG treat-ment, the numbers of CD45þ leukocytes and CD45� non-leuko-cytes were unchanged from day 1 to day 7, as expected.
Most importantly for this analysis, MAA beads increased thenumber of macrophages relative toMMbeads at day 7 (Figs. 4D and5D), consistent with the earlier observations of higher expression ofTNFa and IL1b genes in diabetic wounds at the same time point [6].The increase with histological analysis was not statistically signif-icant, presumably because of different regions of interest or highersensitivity of flow cytometry to identify cells with low F4/80expression. For all treatment groups, themean fluorescent intensityof F4/80 increased from day 1 to 7 (Fig. 5E), suggesting increasedmaturation [57].
In contrast to MAA beads, control MM beads were surroundedby a thick layer of macrophages (Fig. 4A); a common observationwith implanted biomaterials [reviewed in Refs. [58,59]]. Similarly, athick layer of cells was observed in Mason's trichrome andCLARITY-processed images around MM but not MAA beads (Fig. 2Aand D). At day 7, MM beads also had a higher density of CD45� cells(Fig. 5B), a majority of which were believed to be fibroblasts.Overall, the distribution of F4/80 staining, the presence of a thicklayer of cells and a higher number of CD45� cells suggested anincreased fibrotic response to control MM beads [60,61], a featureof a conventional foreign body response [62e64]. On the otherhand, the vascular regenerative MAA beads lacked a thick layer ofcells (Fig. 2A and D) and maintained low levels of CD45� cells(Fig. 5B); these are indicative of what we have termed as an alter-native foreign body response.
Since macrophages play an important role in vascularization[65e67], the results presented here supported our premise thatmacrophages were one of the orchestrators of vessel formation
A. Lisovsky et al. / Biomaterials 98 (2016) 203e214212
with MAA beads.
4.4. The effect of MAA beads on macrophage polarization
Macrophage phenotype varies depending on the conditions thathave led to their activation [68]. However, it is still convenient touse a distinction between “M1” and “M2” cells as a simplification ofthe spectrum of polarization states. While primarily considered asclassically activated inflammatory macrophages, M1 macrophageshave a role in initiating vessel formation [60] and M2 macrophagesthat arise later in the foreign body response are involved in pro-moting vessel maturation [60,69,70]. Improved vascularizationcorrelated with increased numbers of M2 macrophages [71];however, exogenous administration of M2 macrophages 1e3 dayspost-injury failed to improve vascularization in a cutaneous woundmodel [72], although this may have reflected changes that occur inpre-polarized macrophages upon implantation. We hypothesizedthat MAA beads polarized macrophages to a M2 state, consistentwith the increased vascularization.
Indeed, treatment with MAA beads induced a M2 macrophagepolarization bias: therewas an increased density of CD206þ cells inproximity to the beads (within 200 mm) in females but not males atday 4 (Fig. 4CeE). An increase in CD206þ cell density was sug-gested in males at day 7 but no difference was noted in females.These results highlighted the sex differences in macrophage po-larization observed by others [73,74] and may have been an un-derlying reason for differential MAA-mediated vascularizationresponse between males and females (Fig. 2B and C).
Flow cytometry analysis allowed quantification of macrophagesthat were CD206þ, but additionally allowed for discriminationbetween those that were also MHCIIþ or MHCII�. TermingMHCIIþCD206� cells as M1 cells and MHCII�CD206þ cells as M2,at day 7, MAA beads increased the density of M2 cells fourfoldcompared to MM beads (Fig. 6C), while MM beads induced a nearlynine-fold increase in M1 macrophages relative to MAA beads(Fig. 6D). Treatment with both controls (MM beads and PEGvehicle) elicited a more inflammatory macrophage response, withhigher numbers of M1 or double positive MHCIIþCD206þ macro-phages by day 7, relative to MAA beads (Fig. 6E and F). We presumethat these double positive cells are those in transition from theinitial inflammatory M1 cells to the later M2 cells, but additionalresearch is required to understand the role of these “hybrid”macrophages in vascularization.
While it is evident that MAA beads induced a bias in macro-phage polarization towards M2, it is unclear why this happens.Several interconnected mechanisms are likely involved and studiesare underway to clarify these mechanisms. Several hypotheses areproposed here. In this subcutaneous injection model, it was notedthat deliberate nicking of small vessels enhanced the vascularregenerative effect of MAA beads (but not MM beads), suggesting arole for whole blood or one of its components. Separately, we haveseen that incubating beads with plasma or serum resulted in morecomplement proteins (e.g., C1q, Factor H) adsorbed to MAA than toMM beads; yet complement was activated to a lower degree withMAA beads [55]. C1q has been implicated in macrophage polari-zation [75,76] and its differential adsorption to MAA beads may inpart explain the polarization bias. Alternatively, the increasedneutrophil density in MAA-treated mice (Fig. 5C) likely led to moreapoptotic neutrophils, which has also been implicated in M2 po-larization [77,78].
Finally, Shh expression was increased by MAA beads in macro-phages (Supplementary Information, Fig. 4), suggesting a link be-tween Shh and M2 polarization with MAA beads. Exposure ofprimary macrophages and a macrophage cell line (RAW264.7 cells)to a pathogen upregulated markers of alternatively activated M2
macrophages (Arg1, Fizz1 and Ym1) and this response was inhibitedwith Shh pathway specific antagonists [13]. Primary brain tumorsexpressing Shh were characterized by increased infiltration ofmacrophages and expression of genes implicated in polarization ofmacrophages towards M2 [55]. Thus, it may be that the Shhpathway has been activated during the initial wave of inflammationwhich then served as a M2 polarization signal [54,79].
4.5. The insight into mechanism of vascular regenerative MAAbeads
The results described here and previously [reviewed in Ref. [2]]revealed that MAA-based biomaterials elicit its regenerativeproperties by modulating several aspects of vascular biology. Wehypothesize that upon interaction with tissue, MAA-based bio-materials differentially adsorb proteins including complement (e.g.,C1q) [56], which then modulate phosphorylation pathways withinminutes of contact between the biomaterial and cells [14]. Subse-quently, differential expression of mRNA [6e8] and proteins lead toa modified initial inflammatory response characterized byincreased neutrophil infiltration. This in turn results in the activa-tion of other signaling pathways (i.e., Shh) and the modulation ofcells involved in vascularization (i.e., macrophages and endothelialcells). Follow up studies will need to link these components. Forexample, whether complement, neutrophils or Shh signaling leadsto vascularization by modulating yet other cells and pathways re-mains to be elucidated. It is clear that MAA-based biomaterialsactivate a complex network of events to effect vascularregeneration.
What is not clear is how all of this from protein adsorptionthrough Shh pathway modulation to vascularization is determinedby the properties of the biomaterial. We attribute the vascularregenerating effect to themethacrylic acid and its strong charge buthow the properties of this charge (e.g., pKa) influence the adsorbedprotein and whether other similar anionic polymers would also bevascular regenerating is an as of yet unanswered question.
5. Conclusion
MAA beads improved vascularization in healthy mice of bothsexes after subcutaneous injection. The higher vessel density wasaccompanied by an increase in the expression of GFP (Shh) and b-Gal (Ptch1), differences in inflammatory cell infiltration (includingmore neutrophils at day 1 and macrophages at day 7) and amacrophage polarization bias towards M2. These results suggestthat the Shh signaling pathway and an altered inflammatoryresponse are associated, and together may be involved in the MAA-mediated modulation of the host response and its beneficialvascular regenerative effect. This subcutaneousmodel may prove tobe a useful tool for further understanding of the host response tobiomaterials.
Acknowledgments
The authors acknowledge financial support from the OntarioResearch Foundation and the Natural Sciences and EngineeringResearch Council (NSERC). A. Lisovsky acknowledges scholarshipsupport from the Province of Ontario, the University of Toronto andthe NSERC Collaborative Research and Training Experience(CREATE) in Manufacturing Materials and Mimetics (M3) trainingprogram. D. K. Y. Zhang acknowledges scholarship support from theProvince of Ontario, the University of Toronto and the CanadianInstitutes for Health Research. The authors acknowledge help fromI. Talior-Volodarsky and R. Mahou in the setup of the in vitromacrophage experiment and C. Lo for his surgical expertise.
A. Lisovsky et al. / Biomaterials 98 (2016) 203e214 213
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2016.04.033.
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[79] W.J. Zacharias, X. Li, B.B. Madison, K. Kretovich, J.Y. Kao, J.L. Merchant, et al.,Hedgehog is an anti-inflammatory epithelial signal for the intestinal laminapropria, Gastroenterology 138 (2010) 2368e2377.
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80
S17. Curriculum vitae.
Page 1 of 4
DAVID K.Y. ZHANG
Phone
+1 (647) 974-4510
Email [email protected]
EDUCATION
University of Toronto, Toronto, Ontario, Canada
Institute of Biomaterials and Biomedical Engineering (IBBME)
MASc in Biomedical Engineering
Advisor: Prof. Michael V. Sefton
GPA: 4.0/4.0
Sept 2014 – Jun
2016
McGill University, Montréal, Canada
BSc in Honours Immunology
Advisor: Prof. David Juncker
GPA: 3.93/4.0
Sept 2011 – Apr
2014
RESEARCH
MASc Thesis
Institute of Biomaterials and Biomedical Engineering, University of Toronto,
Canada
Prof. Michael Sefton
Inflammatory cell responses to vascular regenerative methacrylic acid-containing
materials.
Sept 2014 – Jun
2016
Undergraduate Thesis Department of Biomedical Engineering, McGill University, Montréal, Canada
Prof. David Juncker
Assessing cytokine responses in stressed microglia cells.
Sept 2013 – Apr
2014
Research Assistant Department of Biomedical Engineering, McGill University, Montréal, Canada
Prof. David Juncker
Development of an automated microfluidic device for ultrahigh-sensitive
quantification of biomarkers.
May 2013 –
Aug 2013
Research Assistant Faculty of Medicine, McGill University, Montréal, Canada
Dr. Ivan Rohan, MD
Development and implementation of an E-learning oncology course for primary
care physicians.
May 2012 – Jan
2013
D.K.Y. Zhang
Page 2 of 4
Research Assistant Ecology Lab, Faculty of Environment, University of Waterloo, Canada
Kari Olsen
Monitoring soil content, water quality, aquatic specimens in the Region of
Waterloo.
Sept 2009 – Jun
2011
AWARDS
SGS Conference Grant. University of Toronto. $880.
May 2016
Canadian Graduate Scholarship- Master’s. Canadian Institute of Health
Research (CIHR). $17,500.
Sept 2015
Institute of Biomaterials and Biomedical Engineering (IBBME) Graduate
Fellowship. University of Toronto. $2,000.
Sept 2015
NSERC CREATE Trainee in Manufacturing, Materials, and Mimetics (M3).
University of Toronto. $15,000. Stipend declined.
Aug 2015
Barbara and Frank Milligan Graduate Fellowship. University of Toronto.
$5,311.
Aug 2015
Queen Elizabeth II & Thomas Noakes Scholarship in Science and Technology.
University of Toronto. $15,000.
Aug 2014
Canadian Graduate Scholarship- Master’s. Natural Sciences and Engineering
Research Council of Canada (NSERC). $17,500. Declined.
Aug 2014
Dean’s Honour List. McGill University (All three years).
May 2014
Undergraduate Research Award. Natural Sciences and Engineering Research
Council of Canada (NSERC). $5600.
May 2013
Alma Mater Scholarship. McGill University. $3000.
Quebec University Support Bursary. McGill University. $1000.
National AP Scholar. Canada. $50.
Sept 2011
Sept 2011
Sept 2011
PUBLICATIONS
1. A. Lisovsky*, D.K.Y. Zhang*, M.V. Sefton, “Effect of methacrylic acid beads on the sonic
hedgehog signaling pathway and macrophage polarization in a subcutaneous injection mouse
model”, Biomaterials, 2016. doi:10.1016/j.biomaterials.2016.04.033
* These authors contributed equally.
D.K.Y. Zhang
Page 3 of 4
PRESENTATIONS
I. Tailor-Volodorsky, D.K.Y. Zhang, R. Mahou, M.V. Sefton. The effect of methacrylic acid-
stimulated macrophages on endothelial cells. Poster. World Biomaterials Congress. Montreal, Canada.
May, 2016. DOI: 10.3389/conf.FBIOE.2016.01.02635
D.K.Y. Zhang, M.V. Sefton. Methacrylic acid- containing beads modulate macrophage polarization in
a vascularizing subcutaneous mouse model. Oral Presentation. World Biomaterials Congress.
Montreal, Canada. May, 2016. DOI: 10.3389/conf.FBIOE.2016.01.01325
K. Zhang, M.V. Sefton. Macrophages are polarized to a CD206+MHCII- phenotype by methacrylic
acid-containing beads in a subcutaneous injection model. Poster. Institute of Biomaterials and
Biomedical Engineering Scientific Day, University of Toronto. Toronto, Canada. May, 2015.
K. Zhang, G. Zhou, A. Ng, D. Juncker. Optimizing gold nanoparticle generation for quantitative
plasmonic ELISA. Poster. Biomedical Engineering Symposium, McGill University. Quebec, Canada.
September, 2013.
TEACHING
Head Lab Teaching Assistant
BME 205, Biomolecules and Cells.
Division of Engineering Science, Faculty of Arts & Science, University of Toronto.
Jan 2016 – Apr
2016
Teaching Assistant BME 395, Biomedical Systems Engineering II: Cells and Tissues.
Division of Engineering Science, Faculty of Arts & Science, University of Toronto.
Sept 2015 –
Dec 2015
Lab Teaching Assistant BME 205, Biomolecules and Cells.
Division of Engineering Science, Faculty of Arts & Science, University of Toronto.
Jan 2015 – Apr
2015
MENTORSHIP
Mohammad Saleh, Thesis Student.
Division of Engineering Science, Faculty of Arts & Science, University of Toronto.
Sept 2015 – Apr
2016
Robert Brais, Thesis Student.
Department of Electrical Engineering, Faculty of Engineering, McGill University.
Sept 2013 –
Aug 2014
COMMUNITY
Student mentor. Saturday Day Program, University of Toronto.
Content development committee. Stem Cell Talks 2016, Let’s Talk Science.
Graphics director. IBBME Scientific Day, University of Toronto.
Jan 2016 – Apr 2016
Sept 2015 – Mar 2016
Sept 2015 – Mar 2016
D.K.Y. Zhang
Page 4 of 4
Judge recruitment committee. IBBME Scientific Day, University of Toronto.
Science Rendezvous volunteer. Toronto, Ontario.
Tech Staff. Canadian Biomaterials Society Conference. Toronto, Ontario.
Student mentor. Saturday Day Program, University of Toronto.
Graphic design committee. IBBME Scientific Day, University of Toronto.
Content development committee. Stem Cell Talks 2015, Let’s Talk Science.
Let’s Talk Science volunteer. Toronto, Ontario.
University of Toronto peer tutor (chemistry, biochemistry, immunology).
McGill Sketching Club VP. McGill University.
CaPS tutor (calculus, immunology, organic chemistry). McGill University.
Volunteer at the Montreal General Hospital
Peer tutor (physics, chemistry, calculus), Waterloo, Canada
Art instructor at Little Artists Workshop, Waterloo, Canada
Sept 2015 – Mar 2016
May 2015
May 2015
Jan 2015 – May 2015
Sept 2014 – Mar 2015
Sept 2014 – Mar 2015
Sept 2014 – Jun 2016
Sept 2014 – Jun 2016
Sept 2012 – May 2014
Sept 2013 – May 2014
Jan 2012 – Apr 2012
2007 – 2011
2007 – 2011