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Administered out of Wake Forest Institute for Regenerative Medicine Lessons from Translation in Biomaterials Design J. Elisseeff, PhD Morton Goldberg Professor and Director of the Translational Tissue Engineering Center, Department of Biomedical Engineering and Wilmer Eye Institute, Johns Hopkins University AABB 2018 Boston Elsie Russell's Prometheus The beautiful fables of the Greeks, being proper creations of the imagination and not of the fancy, are universal verities. What a range of meanings and what perpetual pertinence has the story of Prometheus!-- Ralph Waldo Emerson, "Essays" Lessons from translation Hillel et al., Science Translational Medicine, 2011 Kythera Biopharma Pilot clinical testing: pre-abdominoplasty 1.2 * * 1.0 Photofiller 0.8 0 /t x 0.6 Height t Height 0.4 volume ratio volume 0.2 0.0 Filler PEG100-HA24 HA24 PEG100-HA20 HA20 Ligh Ctrl Ligh Ctrl t t COI: Kythera Local Environment affects biomaterial responses (in people and animals) DERMIS ADIPOSE Hillel et al., Science Translational Medicine, 2011 What are important cells in the biomaterials response? Location, location, location….... CD4+ T Cells Implant Tissue ECM array Beachley, Wolf, Sadtler et al., Nature Methods, 2015 Can we learn more about the impact of the local environment? Beachley, Wolf, Sadtler et al., Nature Methods, 2015 Structure and Composition of ECM array Lung ECM: ECM-Associated: Collagens ECM Glycoproteins 10,000 Adipose Proteoglycans ECM Regulators Bladder ECM-affiliated Proteins Secreted Factors Bone 1,000 Adipose Bladder Bone Brain Kidney Brain Cartilage 100 Cardiac Kidney 10 Liver Cartilage Cardiac Spleen Liver Sm. Intestine PSMs Normalized Lung 1 SI ECM ECM-Associated Non-ECM Spleen Beachley, Wolf, Sadtler et al., Nature Methods, 2015 2.0 Skin Lung Breast Fibroblast 1.5 r e b m u 1.0 N l l e C 0.5 0.0 r e n -I e l e c y r g n e e n i l g ro s a e e n e n d o ra o a t o i n iv e ti d C il n p rd d L u l s la B B rt o i a i L p e a C d C K S t B A In C . m S Cancer Cells Cancer Cell Adhesion B16 0.8 -Melanoma Adhesion Breast Cancer Proliferation 0.6 *Bo, Ad 0.4 Doubling0.2 Rate (1/day) 0 *Bo, *Bo Br, Ad, Lg, Lv Stem Cells *Bo Beachley , Wolf, Sadtler Parent et al., Metastatic Nature Methods, 2015 Flow cytometry In vitro macrophage- material analysis Gene expression What are the mechanisms of tissue repair with biological scaffolds? Tissue/ECM-derived Materials 1. T H Petersen et al. Science 2010; 329:538-5411. 2. H T Ott et al. Nature Medicine 2008; 14:213–221 3. DermaMatrix, Synthes, Inc. 2. Grafton® DBM, Osteotech, Inc. Tissue-derived materials or ECM scaffolds Particles Hydrogels Vitrified Cardiac Tissue Grafts Native Decell Vitrified SIS UBM Cornea The immune system: first responder to materials Synthetic Natural Sadtler et al., Science, 2016 Innate and Adaptive Immune System are Intimately Linked Innate Immunity Adaptive Immunity Fast, less specific Infected Slower, very specific Tumorcell MaterialsTrauma Antigen Presenting Cell (APC) Dendritic Cell NK Cell Macrophage Costimulation MHC Neutrophil TCR Macrophage T Cell B Cell Biological (ECM) scaffolds repair muscle defects Scaffold (C-ECM) IL-4 Expression No Treatment 6 weeks Ferrand BK, Kokini K, Badylak SF, Geddes LA, Hiles MC, Morff RJ. Directional porosity of porcine small-intestinal submucosa. Journal of biomedical materials research. 1993;27:1235-41. Which immune cells enter the wound and biomaterial? Dendritic Cells F4/80MACS+CD11c- CD11c+F4/80- + - CD11c+F80- 12 F480 CD11c 1.2 (35.2%) ) Saline 5 0 Collagen 1 8 0.8 x ( B-ECM t n C-ECM u o C l 4 0.4 l e C 0 0.0 0 2 4 6 0 2 4 6 1.5 0.08 T cells B cells CD3+ + CD19+ CD3 + ) CD19 5 0 0.06 1 1.0 x ( t n 0.04 u o C l 0.5 l (3.8%) e 0.02 C 0.0 0.00 0 2 4 6 0 2 4 6 Weeks Post-Operation Kaitlyn Sadtler et al.,Science, 2016 Macrophages are very complex Adaptive immune system required 12 WT 80 **** * Rag-/- 60 ) 10 * 2 10 0 1 for macrophage responsesx to ECM 8 ( 8 0 0 7 6 6 F A 6 4 8 4 D C 2 2 0 0 Saline Collagen Bone Cardiac Saline Collagen Bone Cardiac CD86 **** CD206 **** 80 80 12 WT 80 **** **** * Rag-/- 60 ) ) 10 2 * 2 10 0 0 60 60 1 1 x x 8 ( ( 8 C 0 **** * ** 0 P 40 40 7 6 *** A 6 F 6 A 0 2 6 4 8 4 D 20 20 D C C 2 2 0 0 0 0 Saline Collagen Bone Cardiac Saline Collagen Bone Cardiac Saline Collagen Bone Cardiac Saline Collagen Bone Cardiac **** **** 80 80 **** ) 2 0 60 60 1 x ( C **** * ** P 40 40 *** A 6 0 2 D 20 20 C 0 0 Saline Collagen Bone Cardiac Saline Collagen Bone Cardiac No T or B cells The Adaptive Immune System: T cells in the wound bed and ECM T cell Subsets in Implant/Wound **** **** 100 100 ** * Saline ** ) **** * Collagen ) % 80 ( B-ECM **** % 80 + ( C-ECM 3 + D 3 60 C D 60 f C o f t * o 40 n t * 40 e n * c e r * c e 20 r P e 20 P 0 0 CD4+ CD8+ CD4+ CD8+ CD4+ CD8+ CD4+ T cell complexity and function Tumor Intracellular pathogen Autoimmunty Th1 - Influences local tissue IL12 immune and stromal cells TCR IL4 Helminth Allergy - Systemic impact potential Regeneration + CD4 T IL6 Th2 - Antigen specificity Cell IL1b TGFb Extracellular pathogens IL17a,f Autoimmunity Fibrosis IL22 Th17 + 1000 Collagen CD3+ TranscriptomeCD3 Transcriptome ) t C B-ECM 8 Δ 100 Δ C-ECM - 8 2 ( 4 e F 10 n i l A a S 1 3 o t D Q 0.1 C R 0.01 5 1 4 a d b 1 l 4 1 7 8 g s 9 4 n 2 5 2 4 5 1 8 f g 1 2 a 4 c 1 a 3 0 3 2 4 Il a l it s a d l o f 1 Il s 1 r 1 2 1 n n 2 2 7 rf r b r p 1 a Il d d 8 b a l 2 2 0 c rs f lm c l c l l l T f x l I o f 2 x l t g 1 C d B S F t c d d 4 I f s o c x I I I I I l1 g Il I a a d C C C d C C d n f G x C b I R T o J C P T n S C T F G F4/80 PE/Cy7 C T NKT Th CTL - ve Activation/Proliferation + ve IL2 Inflamm. Th1 Th17 Treg Th2 IFNg Production - Th1 20000 Saline r e TH1 - IFNg ECM b PCL m 15000 CD4+ u N l l ILC e 10000 C CD8+ e t u l o 5000 s CD3+ b A 0 CD4 CD45 e L Th17 (IL-e17) L Thy1.2 n M n M li C C li C C a E IL1P7 Producation - TEh17 P S S Live CD3 CD8 20000 No Abx Abx Saline r TH17- IL17 ECM e * b * * PCL m 15000 IL17 IL17 u N l l e 10000 C e IFNg IL4 IFNg IL4 t u l o 5000 s b A 0 IL17a IL17a IL17a IL17a L L ECM PC ECM PC IFNg IL4 IFNg IL4 Saline Saline No Abx Abx + 1000 Collagen CD3 Transcriptome ) t C B-ECM Δ 100 Δ C-ECM - 2 ( e 10 n i l a S 1 o t Q 0.1 R 0.01 5 1 4 a d b 1 l 4 1 7 8 g s 9 4 n 2 5 2 4 5 1 8 f g 1 2 a 4 c 1 a 3 0 3 2 4 Il d d 8 a l it s a d 2 2 l o f 1 Il s 1 r 1 2 1 n n 2 2 7 rf r b r p 1 a g Il 1 d b S a tl c d d 0 c rs f lm c l c Il Il Il T If x Il 1 I o f l2 x Il t a d C B C F d 4 I f fs o c x b Il R g I o a J C C C C d n n G S x C T T F G C P C T T C NKT Th CTL - ve Activation/Proliferation + ve IL2 Inflamm. Th1 Th17 Treg Th2 CD3 AF488 F4/80 PE/Cy7 What are T cells doing in the wound/biomaterial? CD3+ Transcriptome mTOR signaling pathway Environmental sensor and regulator for cell metabolism, proliferation, survival Deptor Rheb Protor-1 PRAS40 Deptor mLSTB mTOR mLSTB mTOR mSIN1 Rapto Rictor r Th1 mTORC1 mTORC2 Th2 Jonathan Powell Th2 T cells are Critical in Shaping the Regenerative Microenvironment Fibrosis Minimal Fibrosis Adipose Actively Remodeling Intra-muscular Adipose Fibrosis Small Fibers Rescue: CD4+ Wild Type No Rescue: CD4+ Th2 Deficient Systemic immunological changes Systemic RQ to Saline 15 ** M WT e C n -/- i Rag ) l E l - a a Cd4-/- c S C 10 o L ( l a ** n 500 μm i u **** 1 week 5 g n I * 200 μm 0 (2 4 Interleukin Collagen B-ECM C-ECM Collagen B-ECM C-ECM **** ) 15 l a t s M i e D C n ( i l - l E ΔΔCt a - 10 a i h S C c ) a 3 weeks r B / y 5 500 μm r a l l i x A 0 200 μm Collagen B-ECM C-ECM Collagen B-ECM C-ECM How do ECM scaffolds modulate the immune system? • Macrophages • Dendritic cells • T cells Acellular Adipose Tissue (AAT) • An Off-the-shelf alternative to fat grafting • Cadaveric adipose tissue • Lipid/Cell remnants removed • GMP injectable format Phase I Clinical Trial Design Sample Study Title Population Size Design End Points Follow-up Men & Pilot Clinical IMPLANT EXCISION women, Testing of a Novel 2 x 1 mL (Panniculectomy, 1 – 12 aged 18-50 Treatment Soft Tissue subcutaneous abdominoplasty) weeks years, Group (n=8) Reconstruction injections post- healthy, with Solution per subject 1 – 18 weeks excision no tissue post-injection defects.
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