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1

Supplemental Materials ColonyStimulatingFactor-1Receptorisacentralcomponentoftheforeignbodyresponsetobiomaterialimplantsinrodentsandnon-humanprimatesJoshuaC.Doloff1,2,3,OmidVeiseh1,2,3,4,ArturoJ.Vegas1,2,5,HokHeiTam1,3,ShadyFarah1,2,3,Minglin Ma1,2,6, Jie Li1,2, Andrew Bader1,2, Alan Chiu1,2, Atieh Sadraei1, Stephanie Aresta-Dasilva1,2,MarissaGriffin1,SiddharthJhunjhunwala1,2,MatthewWebber1,3,SeanSiebert1,2,Katherine Tang1,2, Michael Chen1,2, Erin Langan1,2, Nimit Dholokia1,2, Raj Thakrar1,2,Meirigeng Qi7, Jose Oberholzer7, Dale L. Greiner8, Robert Langer1,2,3,9,10,11, and Daniel G.Anderson1,2,3,9,10,11*

1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute ofTechnology,500MainStreet,Cambridge,MA,02139,USA

2.DepartmentofAnesthesiology,BostonChildren’sHospital,300LongwoodAve,Boston,MA02115,USA

3. Department of Chemical Engineering, Massachusetts Institute of Technology, 77MassachusettsAvenue,Cambridge,MA,02139,USA

4.Currentaddress:Sigilon, Inc., 161 First St., Cambridge, MA 02142, USA

5. Current address: Department of Chemistry, Boston University, Boston, Massachusetts,USA

6. Current address:Biological andEnvironmentalEngineering, CornellUniversity, Ithaca,NY14853,USA

7. Division of Transplantation, Department of Surgery, University of Illinois at Chicago,Chicago,IL

8.PrograminMolecularMedicine,UniversityofMassachusettsMedicalSchool,Worcester,MA01605,USA

9. Division of Health Science Technology, Massachusetts Institute of Technology, 77MassachusettsAvenue,Cambridge,MA,02139,USA

10. Institute forMedicalEngineeringandScience,Massachusetts InstituteofTechnology,77MassachusettsAvenue,Cambridge,MA,02139,USA

11. Harvard-MIT Division of Health Science and Technology, Massachusetts Institute ofTechnology,77MassachusettsAvenue,Cambridge,MA,02139,USA

*email:[email protected];Tel.:+16172586843;fax:+16172588827.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NMAT4866

NATURE MATERIALS | www.nature.com/naturematerials 1

http://dx.doi.org/10.1038/nmat4866

2

Supplemental Index of Sections 1) Supplementary Table 3

Supplemental Table S1 – Knockouts and targeted depletion fibrosis summary. Supplemental Table S2 – Negative endotoxin results for all materials used in thisstudy. Supplemental Table S3 – Mouse (m) (host) forward and reverse primer sets used for qPCR analysis of RNA levels.

2) Supplementary Figures 6 Supplementary Figure S1 – The same immune responders adhere to implanted biomaterial alginate spheres in both the IP and subcue (SC) sites. Supplementary Figure S2 – Kinetic profiling of 30 cytokines in the blood shows no global response to alginate. Supplementary Figure S3 – Photo sequence for retrieval process. Supplementary Figure S4 – Additional kinetic expression profiling for immune-related factors associated with fibrotic cascade. Supplementary Figure S5 – Complete panel of phase contrast images for knockouts. Supplementary Figure S6 – Additional B cell WT and (IghMnull) knockout model characterization, for reduced B cell response and corresponding reductions in fibrosis. Supplementary Figure S7 – Additional histology (H&E and Masson’s Trichrome staining) panels for subcutaneously implanted wildtype and knockout mice. Supplementary Figure S8 – Additional FACS characterization of mock, distant, and implanted subcue sites. Supplementary Figure S9 – Complete panel of phase contrast images for targeted, serial innate immune depletions (as shown in Figure 4). Supplementary Figure S10 – Flow analysis plots, comparing wildtype versus targeted innate immune depletion responses to implantation of alginate. Supplementary Figure S11 – Flow analysis plots, showing specificity of serial innate immune depletions. Supplementary Figure S12 – Complete NanoString analysis for identification of inflammation and immune population-specific factors. Supplementary Figure S13 – CSF1R inhibition prevents fibrosis of IP implanted alginate spheres (cont.) Supplementary Figure S14 – Macrophage elimination or, minimally, CSF1R inhibition prevents fibrosis of IP implanted 500 µm glass ceramic spheres. Supplementary Figure S15 – Macrophage elimination or, minimally, CSF1R inhibition prevents fibrosis of IP implanted 500 µm polystyrene polymer spheres. Supplementary Figure S16 – Complete NanoString analysis for cytokine signaling common to host responses across multiple material classes. Supplementary Figure S17 – Macrophage-associated VEGF production lost upon macrophage depletion is instead spared with CSF1R inhibition. Supplementary Figure S18 – CSF1R inhibition leaves many macrophage functions intact. Supplementary Figure S19 – FACS plots and phase contrast images showing effects of CXCL13 antibody neutralization on B cell recruitment and downstream fibrosis. Supplementary Figure S20 – Additional primate histology and immunofluorescence panels showing similar foreign body responses.

3. Supplementary References 29





3

1. Supplementary Tables:

Table S1. Knockouts and targeted depletion fibrosis summary. Serial or combined immune

perturbations were used across various C57BL/6 strains to determine which cell populations are

necessary for immune-mediated fibrosis. After extensive characterization, macrophages, and not

neutrophils, are the only cell population required for downstream fibrotic sequestration of

implanted alginate spheres. Right column: representative summary responses based on phase

contrast images showing fibrosis levels on 500 µm alginate spheres retrieved from wild type

C57BL/6 mice (n = 5/group), after 14-day intraperitoneal implantations. *, as reported. N/A,

while available, not tested due to not being essential players.

Background Mutation Tcelldeficient

Bcelldeficient

NKcelldeficient

Additionaldeficiencies

FibrosisStatus

C57BL/6 None - - - - ✓,Fibrosed

C57BL/6 NOS2 - - - MacroslackiNOS

N/A

C57BL/6 C3 - - - NoComplement

✓,Fibrosed

C57BL/6 IL15 - - + Otherimmunedysregulation

N/A

C57BL/6 Nude + - - - ✓,Fibrosed

C57BL/6 muMT - + - - ✓,ê

C57BL/6 Rag2 + + - - ✓,MostlyFibrosed

C57BL/6 Rag2/IL2rg + + + Macrodysfunction

✓,NoFibrosis

C57BL/6 MAFIA* - - - Macrodepletion

✓,NoFibrosis1

C57BL/6 α-MΦ - - - Macrodepletionorinhibition

✓,NoFibrosis✓,NoFibrosis

C57BL/6 α-Neutrophil

- - - Neutrodepletion

Fibrosis

C57BL/6 α-MΦ&α-Neutrophil

- - - Macro&Neutro

depletion

✓,NoFibrosis

C57BL/6 α-CXCL13 - + - LackofBcells ✓,ê





4

Table S2. Negative endotoxin and glucan results for all materials used in this study. As

determined by Charles River Labs sample submission, as well as in-house testing, for bacterial

pyrogen and endotoxin. Specifically, E. coli and Limulus Amebocyte Lysates were used as

positive controls to test for the presence of general endotoxin. BDL = below detectable limits.

These negative results have also been corroborated by others in our group, having now been

published in multiple studies1, 2, 3.

Sample Endotoxin Test Glucan Test

Saline control < 0.05 EU/mL (BDL) <10 rg/ml (BDL)

SLG20 alginate 500

µm spheres

< 0.05 EU/mL (BDL) <10 rg/ml (BDL)

SLG20 alginate

solution

< 0.05 EU/mL (BDL) <10 rg/ml (BDL)

Glass spheres < 0.05 EU/mL (BDL) <10 rg/ml (BDL)

Polystyrene spheres < 0.05 EU/mL (BDL) <10 rg/ml (BDL)

Table S3. Mouse (m)-specific (host) forward and reverse primer sets used for qPCR analysis of

RNA levels. Gene names are also shown in parentheses.

Gene Primers (5’ to 3’): Sense & Antisense

Mouse Collagen 1a1

(mCol1a1)

Forward: 5’-CATGTTCAGCTTTGTGGACCT-3’

Reverse: 5’-GCAGCTGACTTCAGGGATGT-3’

Mouse Collagen 1a2

(mCol1a2)

Forward: 5’-GCAGGTTCACCTACTCTGTCCT-3’

Reverse: 5’-CTTGCCCCATTCATTTGTCT-3’

Mouse Alpha Smooth

Muscle actin (mActa2)

Forward: 5’-CGCTTCCGCTGCCCAGAGACT-3’

Reverse: 5’-TATAGGTGGTTTCGTGGATGCCCGCT-3’

Mouse Inflammation

marker Transforming

Growth Factor beta1

(mTGFb1)

Forward: 5’-CCTGAGTGGCTGTCTTTTGAC-3’

Reverse: 5’-ACAAGAGCAGTGAGCGCTGAAT-3’

Mouse Macrophage

marker F4/80 (mEmr1)

Forward: 5’-GATACAGCAATGCCAAGCAGT-3’

Reverse: 5’-TTGTGAAGGTAGCATTCACAAGTGTA-3’





5

Mouse Macrophage

marker CD68 (mCd68)

Forward: 5’-GCCCGAGTACAGTCTACCTGG-3’

Reverse: 5’-AGAGATGAATTCTGCGCCAT-3’

Mouse Myeloid cell

marker CD11b

(mItgam)

Forward: 5’-CCAAGAGAATGCAAAAGGCTTT-3’

Reverse: 5’-GGGGGGCTGCAACAACCACA-3’

Mouse neutrophil

marker Gr1 (mLy6g)

Forward: 5’-TGCCCCTTCTCTGATGGATT-3’

Reverse: 5’-TGCTCTTGACTTTGCTTCTGTGA-3’

Mouse B cell marker

CD19 (mCd19)

Forward: 5’-GGAAACCTGACCATCGAGAG-3’

Reverse: 5’-TGGGACTATCCATCCACCAGTT-3’

Mouse Dendritic cell

(DC) marker CD74

(mCd74)

Forward: 5’-CCCAGGACCATGTGATGCAT-3’

Reverse: 5’-CTTAAGATGCTTCAGATTCTCT-3’

Mouse Langerhans

Dendritic cell (DC)

marker CD207

(Langerin) (mCd207)

Forward: 5’-GGACTACAGAACAGCTTGGAGAATG-3’

Reverse: 5’-TACTTCCAGCCTCGAGCCAC-3’

Mouse Natural killer

(NK) cell marker

NKp46 (mNcr1)

Forward: 5’-GCAACCCCCTGAAACTGGTA-3’

Reverse: 5’-AAGGTTACCTCAGGCTGTGGATA-3’

Mouse Adaptive helper

T cell marker CD4

(mCd4)

Forward: 5’-GAAGATTCTGGGGCAGCATGGCAAAG-3’

Reverse: 5’-TTTGGAATCAAAACGATCAA-3’

Mouse Cytotoxic T cell

marker CD8a (mCd8a)

Forward: 5’-CTGCGTGGCCCTTCTGCTGTCCT-3’

Reverse: 5’-GGGACATTTGCAAACACGCT-3’

Mouse regulatory T

suppressor cell marker

FoxP3 (mFoxP3)

Forward: 5’-GCCTTCAGACGAGACTTGGAA-3’

Reverse: 5’-CTGGCCTAGGGTTGGGCATT-3’

Mouse b-actin (mActB) Forward: 5’-GCTTCTTTGCAGCTCCTTCGTT-3’

Reverse: 5’-CGGAGCCGTTGTCGACGACC-3’





6

2. Supplementary Figures:

Supplemental Figure S1. The same innate and adaptive immune responders adhere to implanted

biomaterial alginate spheres in both the IP and subcutaneous (SC) sites. SLG20 alginate 500 µm

diameter spheres were implanted into the SC space of C57BL/6 mice, where they were retained

for 14 days and analyzed upon retrieval. qPCR analysis of innate and adaptive immune

population and fibrosis markers present in mock implanted, M, versus fibrosed implanted

alginate-embedded subcutaneous tissues, A. Mf = macrophages, DCs = Dendritic cells, NKs =

Natural Killer cells. Data: mean ± SEM, n = 5. qPCR statistical analysis: one-way ANOVA with

Bonferroni multiple comparison correction *: p < 0.05, **: p < 0.001, and ***: p < 0.0001; ns =

not significantly different. Experiment run at least twice.





7

Supplemental Figure S2. Kinetic profiling of 30 cytokines in the blood shows no global

response to alginate. Multiplexed Luminex kinetic profiling of protein production of 30

inflammatory cytokines in the serum of C57BL/6 mice at 1, 4, 7, 14, and 28 days post-

intraperitoneal implantation of 500 µm biomaterial alginate spheres. (a) Fold change numbers of

all 30 cytokines at individual points, relative to protein levels of the mock/non-implanted (NT)

control serum samples. (b-e) Individual kinetic plots of the only 4 cytokines (IL-5, IL-6, G-CSF,

and KC) that showed any significant responses (red squares in (a)) in the blood of implanted

C57BL/6 mice. Responses, however, were transient and gone within 4-7 days post-implantation,

suggesting that these increases were instead surgery related. Error bars, mean ± SEM. n = 5

mice per treatment. Performed at least two times.

a b c

d e

FOLD NT D 1 D 4 D 7 D 14 D 28

IL-1a 1 1 1 1 1 1

IL-1b 1 1 1 1 1 1

IL-2 1 1 1 1 1 1

IL-3 1 3 1 2 2 2

IL-4 1 1 1 1 1 1

IL-5 1 12 1 1 2 1

IL-6 1 16 3 1 1 3

IL-10 1 1 2 2 1 2

IL-12(p40) 1 1 1 1 1 1

IL-12(p70) 1 1 1 1 1 1

IL-13 1 1 1 1 2 1

IL-17 1 1 1 1 1 1

Eotaxin 1 1 1 1 1 1

G-CSF 1 24 3 1 1 4

GM-CSF 1 2 1 2 1 1

IFN-γ 1 1 1 1 1 1

KC 1 5 1 1 1 1

MCP-1 1 1 1 1 1 1

MIP-1a 1 1 1 1 1 1

MIP-1b 1 1 1 2 1 1

RANTES 1 1 1 1 1 1

TNF-α 1 2 1 1 1 1

IL-15 1 1 1 1 1 1

IL-18 1 2 1 1 1 2

FGF-basic 1 1 2 3 2 2

LIF 1 1 1 1 1 1

M-CSF 1 1 1 1 1 1

MIG 1 1 1 1 1 1

MIP-2 1 2 1 1 1 1

VEGF 1 1 1 1 1 1

P<0.001

P<0.01

P<0.001

P<0.01





8

Supplemental Figure S3. Photo sequence for retrieval process. 500 µm diameter alginate

microspheres are retrieved from the intraperitoneal (IP) space of wildtype C57BL/6 mice

following a 2-week implantation. 1-2) Incisions were made first into the skin and then the

underlying peritoneum. 3-4) Once the skin and peritoneal wall were successfully resected, the

intestines were moved to the side exposing both the IP omental and epididymal fat pads. 5) It

has been consistently and reliably observed that alginate spheres are fibrosed to non-collagen-

encapsulated fat pad tissues (omental, top blue inset square; and epididymal, bottom left and

right inset squares) within the IP space (and never to tissues with collagen capsules, such as the

liver, kidneys, etc.). Implanted materials suffer immune attack from innate and adaptive immune

cell responses that extravasate out of these microvessel rich fat pad tissues1. 5a & 5b) Large

groups of fibrosed alginate microspheres are found around and under both the left and right

epididymal fat pads. Black arrows, fibrosed alginate microsphere(s). 6) While numerous

microspheres are fibrosed directly to and embedded within fat pad tissues, with more adherence

over extended implantation times, many individually fibrosed microspheres can be flushed out of

1 2 3 4

5

5a 5b

6 6b





9

the IP space following a 2-week implantation (6b). Images, representative across all mice;

observed for countless (at least 100-200+) different implantations over the past 4-5 years.

Supplemental Figure S4. Additional kinetic expression profiling for immune-related factors

associated with fibrotic cascade. qPCR analysis (Macrophage marker CD68 (a) and

Transforming growth factor-beta (TGFb1) (b)) on either adjacent fibrosed tissue (yellow) or

alginate spheres alone (blue), relative to day 1 for either tissue or spheres, respectively, showing

that there are similar kinetic responses between surrounding fibrosed tissue and embedded,

fibrosed 500 µm biomaterial alginate spheres, implanted in the IP space of C57BL/6 mice. (c)

qPCR showing no significant changes in IP tissues taken 1, 4, 7, 14, or 28 days post-mock

(saline) implant; Transforming growth factor-beta (TGFb1) and Tumor necrosis factor-alpha

(TNFa) are both graphed relative to day 1. Error bars, mean ± SEM. n = 5 mice per treatment.

Experiments were performed twice. qPCR statistical analysis: one-way ANOVA with

Bonferroni multiple comparison correction *: p < 0.5, **: p < 0.001, and ***: p < 0.0001, vs

non-implanted (N) controls. M = mock-implanted, and N/A = not applicable, for spheres alone.

(d) Western blotting time course for chemokine CXCL13 expression in mock implant tissue vs.





10

day 1, 4, 7, 14, and 28 tissue and sphere protein samples retrieved from the intraperitoneal space

of C57BL/6 mice, showing similar kinetics to Cxcl13 gene expression as shown by Nanostring

analysis in Figure 2d. Run at least twice.

Supplemental Figure S5. Complete panel of phase contrast images for all knockouts. Samples,

retrieved Ba-crosslinked SLG20 spheres of 500 µm diameter spheres implanted into the

intraperitoneal space of wildtype (a) and various adaptive and/or innate immune compromised

B"KO"SLG20"

T"&"B"KO"SLG20"

WT"SLG20"

Rag2/ϒ"SLG20"

a

b

c

d

T"KO"SLG20"

e

f C3"KO"SLG20"





11

knockout (IghMnull, B cell deficiency, (b); Rag2null, T and B cell deficiency, (c); Rag2null/IL2rgnull,

T, B, NK cell deficiency, and Mf and DC dysfunction, (d); T cell knockout (T KO) nude (e) and

complement (C3) knockout (f)) C57BL/6 mice. Fibrosis, as compared to wildtype (WT) control,

was partially decreased upon losing adaptive B cells (B KO), increased when T cells were also

removed (TB KO), and gone completely with additional Mf dysfunction (Rag2/g). Fibrosis was

also not significantly affected by T cell loss alone, nor dependent on C3 complement immune

recognition. Images obtained from all spheres retrieved from individual mice (n = 5/group). The

same material volume of hydrogel spheres was implanted into each mouse in all cases.

Experiments were repeated twice.

Supplemental Figure S6. Additional B cell WT and (IghMnull) knockout model characterization,

for reduced B cell response and corresponding reductions in fibrosis. (a) Additional in vivo

intravital imaging of adaptive B cell behavior and accumulation at day 14 post-implant for

SLG20 sphere implanted C57BL/6-Ccr6 (EGFP) mice (extra images from those shown in Figure

1g showing B cell responses, extravasation from surrounding IP epididymal fat pads and





12

aggregation between quantum dot (pink) encapsulating SLG20 alginate microspheres. (b) IgM

protein levels determined by ELISA for both blood serum (left axis) and IP protein lysates (right

axis) taken from tissue and spheres 14 days post-implant from mock (MT) and implanted

wildtype (WT) and knockout strains ((IghMnull, B cell knockout, B KO; and Rag2null/IL2rgnull

knockout (Rag2/g KO), with T, B, NK cell deficiency, and Mf and DC dysfunction). Loss of

IgM in both blood and IP lysates taken from B cell deficient strains was confirmed. No IgM

increases were detectable in wildtype (WT) responses in the blood but were significantly

increased locally in the intraperitoneal space, suggesting that IgM is present not as secreted

antibody but as a B cell receptor (BCR) on responding B cells. (c) Confocal staining showing

DAPI (cellular nuclei), innate immune macrophage marker CD68 (green), alpha smooth muscle

actin (aSMactin, myofibroblasts, red), B cell marker CD19 (magenta), fluorescent overlay, and

brightfield image for the fibrosis on 500 µm alginate spheres in wildtype (WT) C57BL/6 mice. B

cell CD19 staining was unsurprisingly lost in B cell knockout (B KO) mice. Moreso, however,

confocal imaging confirmed decreased fibrotic overgrowth due to loss of B cells, as seen by

multiple imaging and staining methods (Figure 3). Representative images from n = 3 mice/group

for intravital and n = 5/group for all other analyses. Experiments run at least twice.





13

Supplemental Figure S7. Additional histology (H&E and Masson’s Trichrome staining) panels

for subcutaneously implanted wildtype and knockout mice. Mock (saline injected) (a) and

SLG20 500 µm diameter alginate sphere implanted wildtype (WT) (b), B cell knockout (B KO)

(c), and Mf dysfunctional Rag2null/IL2rgnull (Rag2/g) C57BL/6 mice. 2x and 20x magnifications

are shown in all cases to show both the scale as well as cellular details of varying levels of

fibrosis in each treatment group. Arrows, showing implanted regions surrounding by an outer

fibrosis collagen capsule. *, denote individual fibrosed alginate spheres. As shown in Figure 3,

B cell loss contributes to fibrotic reduction, while Mf dysfunction results in loss of fibrosis

(similar to mock), as compared to WT implanted controls. Representative images from n = 5

mice/group. Experimental analysis run twice.

B"KO"%"SLG20"

WT"%"SLG20"

Rag2/ϒ"%"SLG20"

WT"%"Mock"

20x

2x

20x

2x

*

* *

* * *

*

* *

* * *

*

* *

*

* *

*

*

*

*

*

*

20x

2x

20x

2x

a b

c d





14

Supplemental Figure S8. Additional FACS characterization of mock (left), distant (middle),

and implanted (right) subcutaneous sites. Subcutaneous tissue groups, comparing immune

population compositions of mock implanted (saline injected) to that from alginate-implanted

C57BL/6 mice either at a site distant to (at least 1 cm away) or directly at material delivery

(Implanted). Flow analysis, using specific markers for responding host innate immune

macrophage (CD68+CD11b+) (a), neutrophil (Ly6g/Gr1+ CD11b+) (b), and adaptive B cells

(CD19+IgM+) (c) from dissociated subcutaneous tissue (as percent) 14 days post-subcutaneous

(SC) alginate sphere implantation. Interestingly, macrophage percentage is slightly increased

over that observed on intraperitoneally implantated alginate spheres. Distant tissues, taken from

the same mice implanted SC with 500 µm SLG20 alginate spheres, appear no different than SC





15

tissue taken from mock-implanted (saline injected) mouse controls. N = 5 mice/group.


Supplemental Figure S9. Complete panel of phase contrast images for targeted, serial innate

immune depletions (as shown in Figure 4). Images of retrieved 500 µm diameter SLG20 spheres

following implantation into the intraperitoneal space of wildtype C57BL/6 mice treated with

either saline vehicle (Veh) (a), neutrophil depleting Gr1 antibody (- N) (b), macrophage

depleting clodrosome (- Mf) (c), and both neutrophil and macrophage depletion agents (- Mf &

N) (d). Fibrosis, as compared to vehicle-treated wildtype (WT) controls, was not decreased with

neutrophil depletion (-N), increased when T cells were also removed (TB KO), and gone

completely with additional Mf dysfunction (Rag2/g). Images obtained from all spheres retrieved

from individual mice (n = 5/group). The same material volume of hydrogel spheres was

implanted into each mouse in all cases. Experiments were repeated twice.

a

b

c

d

!"N"

!"MΦ"

Veh"

!"MΦ"&"N"





16

Supplemental Figure S10. Flow analysis plots, comparing wildtype versus targeted innate

immune depletion responses to implantation of alginate. Flow analysis, using specific markers

for responding host innate immune macrophage (CD68+CD11b+) (a), neutrophil (Ly6g/Gr1+

CD11b+) (b), and adaptive B cells (CD19+IgM+) (c) from cells dissociated from fibrosed or non-

fibrosed tissue/spheres as well as spleens (as percent composition) taken 14 days post-

intraperitoneal (IP) implantation, from wildtype C57BL/6 mice treated with either saline vehicle

or macrophage-depleting clodrosome, corresponding to the fibrosis images shown in

Supplemental Figure S9. Interestingly, Mf depletion by clodrosome was specific, leaving

neutrophil responses intact. B cells were also decreased in Mf depleted mice, suggesting that

macrophages are responsible for their recruitment. These results combined with fibrosis images

and data from Figure 3 and Supplemental Figure S9 suggest that neutrophils alone are not

capable of fibrosing biomaterial alginate. Cell population percentages in the spleen (global

immune reservoir) were unaffected by IP-injected clodrosome treatment. N = 5 mice/group.






17

Supplemental Figure S11. Flow analysis plots, showing specificity of serial innate immune

depletions. Specific markers used are for responding host innate immune macrophage

(CD68+CD11b+) (a), neutrophil (Ly6g/Gr1+ CD11b+) (b), and adaptive B cells (CD19+IgM+) (c)

from cells dissociated from fibrosed or non-fibrosed tissue/spheres (as percent composition)

taken 14 days post-intraperitoneal (IP) implantation, from wildtype C57BL/6 mice treated with

either saline (Vehicle, left column), neutrophil depleting Gr1 antibody (- N, middle column), or

both macrophage and neutrophil depletion agents (- Mf & N, right column), corresponding to

the fibrosis images shown in Supplemental Figure S9. All depletion agents proved to be

population specific. Furthermore, neutrophil depletion neither affected macrophage nor B cell

presence on fibrosed spheres, again suggesting their non-importance in a macrophage and B cell

driven fibrotic response. Related, B cells were once again decreased in Mf depleted mice. A

CD11bloGr1lo/-CD68- population, likely repopulating monocytes, was also apparent in Mf

depleted mice. These results combined with fibrosis images and data from Figure 3 and

Supplemental Figure S9 suggest that neutrophils alone are not capable of fibrosing biomaterial

alginate, nor are they required. N = 5/group. Experiments were repeated twice.





18

Supplemental Figure S12. Complete NanoString analysis for identification of inflammation

and (innate and adaptive) immune population-specific factors. Expression of all known mouse

(host) cytokine and cytokine receptors, corresponding to sorted truncated heatmaps in Figures

4d-f for macrophage-specific or associated (downstream) factors, based on removal by depletion

and corroborated by cell sorting. Subsets not affected by CSF1R inhibition suggest altered

macrophage polarization/phenotype and residual function (corroborated by Supplemental Figures

S17-S18). All samples were analyzed from RNA extracts taken from animals 14 days post-

implant in each treatment group, presented on a base 2 logarithmic scale. White, within 2

standard deviations of the mean background of the assay. N = 5/group. Run once.





19

Supplemental Figure S13. CSF1R inhibition prevents fibrosis of IP implanted alginate spheres

(cont.). Complete phase contrast fibrosis images for wildtype C57BL/6 mice treated with either

saline vehicle (Veh) (a) or CSF1R inhibitor GW2580 (b), corresponding to the images in Figure

5d. Images obtained from all spheres retrieved from individual mice (n = 5/group). The same

material volume of hydrogel spheres was implanted into each mouse in all cases. Experiments

were repeated twice.

Vehicle Control

+ CSF1R Inhibitor

a

b





20

Supplemental Figure S14. Macrophage elimination or, minimally, CSF1R inhibition prevents

fibrosis of IP implanted 500 µm glass ceramic spheres. Complete brightfield fibrosis images for

wildtype C57BL/6 mice treated with either saline vehicle (Veh) (a), macrophage depletion agent

clodrosomes (b), or CSF1R inhibitor GW2580 (c), corresponding to the images in Figure 5f.

Images obtained from all spheres retrieved from individual mice (n = 4/group). The same

material volume of hydrogel spheres was implanted into each mouse in all cases. Experiments

were repeated twice.





21

Supplemental Figure S15. Macrophage elimination or, minimally, CSF1R inhibition prevents

fibrosis of IP implanted 500 µm polystyrene polymer spheres. Complete brightfield fibrosis

images for wildtype C57BL/6 mice treated with either saline vehicle (Veh) (a), macrophage

depletion agent clodrosomes (b), or CSF1R inhibitor GW2580 (c), corresponding to the images

in Figure 5f. Images obtained from all spheres retrieved from individual mice (n = 4/group).

The same material volume of hydrogel spheres was implanted into each mouse in all cases.






22

Supplemental Figure S16. NanoString analysis for complete cytokine signaling common to host

responses across multiple material classes. Expression of all known mouse (host) cytokine and

cytokine receptors to identify common inflammation and immune signaling across implanted

hydrogel alginate, ceramic glass, and polymer polystyrene spheres, all retrieved 14 days after IP

implantation into C57BL/6 mice. n = 5/group. Presented on a base 2 logarithmic scale.

Corresponds to excerpted heat map in Figure 5g. Green box in Mock column, within 2 standard

deviations of the mean background of the assay. Nanostring analysis run once.





23

Supplementary Figure S17. Macrophage-associated VEGF production lost upon macrophage

depletion is instead spared with CSF1R inhibition. VEGF Luminex protein quantification for

protein lysates derived from various wildtype and knockout model fibrosed or non-fibrosed

alginate spheres and tissue and retrieved at 14 days. VEGF, important for neovascularization

and wound healing, is significantly reduced in both macrophage depletion groups, but returned to

normal and was not significantly different (ns) than levels observed in fully functional wildtype

(WT) immune competent and implanted control mice following treatment with the CSF1R

inhibitor GW2580 (GW). This finding suggests that residual would healing functions are intact

in CSF1R-inhibited monocyte/macrophages. n = 5/group. Luminex run once. Performed on day

14 samples.





24

Supplemental Figure S18. CSF1R inhibition leaves many macrophage functions intact. a) Skin

incisions (all 1.5 cm in length, rulers are visible on left in all images) were made on day 0, and

then wound clipped shut for both vehicle and daily GW2580-treated C57BL/6 mice (top left).

Wound clips were removed and then replaced each imaging day up until day 7, after which clips





25

were left off completely. By day 7, upon stretching the skin apart, incision sites on GW2580-

treated mice were shut and not pulling apart (top right, red inset). By day 14, there appeared to

be very little scarring in both vehicle and GW2580-treated groups (bottom middle). b) After

both 4 and 14 days, IP immune cells were taken by peritoneal lavage and analyzed by FACS for

innate immune macrophage phenotype (CD68 & CD11b staining). As expected, at both time

points, the mature tissue-resident macrophage phenotype observed in vehicle-treated mice was

shifted (decreased CD68 & CD11b intensities) following daily GW2580 treatment. c) Despite a

phenotype shift, overall cell numbers in the peritoneal exudate were unchanged across untreated,

vehicle-treated, or GW2580-treated groups. d) Confirming visibly healing skin incisions,

histological assessment (H&E and Masson’s Trichrome) show no significant (ns) differences by

width and depth measurements (e) in wound resolution and healing potential between vehicle or

GW2580 treatment groups, by day 14 post-incision; scale bar: 400 µm. f) Peritoneal exudate

macrophages isolated by IP lavage from (n=5) mice in each treatment group were immediately

plated and incubated with fluorospheres for 90 minutes to determine phagocytic activity. Again,

no significant differences were observed between macrophages isolated from vehicle and

GW2580-treated mice. g) Protein lysates were prepared from alginate spheres retrieved 14 days

after IP implantation, and incubation with two different reactive oxygen specie (ROS) substrate

solutions. Once again, no differences in ROS activity were observed between untreated, vehicle-

treated, and GW2580-treated mice. n = 5 mice for all assays. Error bars, mean +/- SE. Run 1-2

times, depending on the assay.





26

Supplemental Figure S19. FACS plots and phase contrast images showing effects of CXCL13

antibody neutralization on B cell recruitment and downstream fibrosis. Flow analysis, using

specific markers for responding host adaptive B cells (CD19+IgM+) from cells dissociated from

fibrosed tissue/spheres (as percent composition) taken 14 days post-intraperitoneal (IP)

implantation, from wildtype C57BL/6 mice treated with either saline (Vehicle) (a) or CXCL13-

neutralizing antibody (- CXCL13). Complete phase contrast fibrosis images for wildtype

C57BL/6 mice treated with either saline vehicle (Veh) (c) or CXCL13-neutralizing antibody (-

CXCL13) (d), corresponding to the images in Figure 5. Images obtained from all spheres

retrieved from individual mice (n = 5/group). The same material volume of hydrogel spheres

was implanted into each mouse in all cases. Experiments were repeated twice.

c

d

Vehicle

- CXCL13





27





28

Supplemental Figure S20. Additional primate histology and immunofluorescence panels

showing similar foreign body responses. H&E (a) and Masson’s Trichrome (b) stained

histological sections of excised IP omentum tissue at 28 days for mock and implanted groups,

showing non-fibrosed fat-laden (no material, Mock) or heavily collagen-deposited and sphere-

embedded omental tissue (Implanted). Corresponds to panels in Figure 6. Magnifications: 10

and 40X. Additional confocal images for immunostained sections from implant alginate sphere

embedded omental tissue excised at 28 days from cynomolgus monkeys. Shown: DAPI (cellular

nuclei), innate immune macrophage marker CD68 (green), and alpha smooth muscle actin





29

(aSMactin, activated myofibroblasts, red; also refer to blue arrows), overlayed together, showing

cellular infiltration around and fibrosis deposition on embedded 500 µm alginate spheres. 5x (c)

and 20x (d) magnifications. Similar to the main panel in Figure 6c. e) Single red channel

images (corresponding to the same images in Figure S20d) showing material-bordering and more

distant punctate aSMactin-staining myofibroblasts (see blue arrows for examples). It should

also be noted, to not be confused, that pericytes covering larger circular blood vessels are also

positive for aSMactin. f) Single channels for DAPI (cellular nuclei), colony stimulating factor-1

receptor (CSF1R) (green), and brightfield views showing high CSF1R staining on both fused

foreign body giants cells (FBGCs) and individual macrophages around embedded 500 µm

alginate spheres. 20x magnification. n = 2 NHPs/group. These experiments were performed

once for SC and twice for IP delivery.

3. Supplementary References:

1. VeisehO,DoloffJC,MaM,VegasAJ,TamHH,BaderAR,etal.Size-andshape-dependentforeignbodyimmuneresponsetomaterialsimplantedinrodentsandnon-humanprimates.Naturematerials2015,14(6):643-651.

2. JhunjhunwalaS,Aresta-DaSilvaS,TangK,AlvarezD,WebberMJ,TangBC,etal.

NeutrophilResponsestoSterileImplantMaterials.PloSone2015,10(9):e0137550.3. VegasAJ,VeisehO,DoloffJC,MaM,TamHH,BratlieK,etal.Combinatorialhydrogel

libraryenablesidentificationofmaterialsthatmitigatetheforeignbodyresponseinprimates.NatBiotechnol2016.





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