Inflammation and wound healing in presence of biomaterials

Inflammation and wound healing in presence of biomaterials Gemma Mestres Applied Materials in Medicine, Department of Engineering Science gemma.mestres@angstrom.uu.se

Outline Why is it important? 1. Immunity and cells involved 2. Inflammation caused by the implantation of a biomaterial 3. Acute inflammation 4. Wound healing in the presence of biomaterials 5. Tailoring immune response of biomaterials 6. Research studies Summary

Why is it important? biomaterial + implanted INFLAMMATORY RESPONSE Tissue healed Surgery by itself will also cause trauma/inflammation Implant fails Inflammatory response can determine the success or fail of a biomaterial

1. Immunity and cells involved

Terminology Immunity Capability of the body to resist disease (specifically infectious disease). Immune system Cells and molecules responsible for immunity. Immune response Collective and coordinated activities against foreign substances. Physiologic function of immune system is to defense against infectious microbes. However, non- infectious foreign substances can also elicit immune responses.

Innate and acquired immunity hours days Innate immunity (nonspecific) Granulocytes and monocytes Provides early line of defense against microbes; respond rapidly to infection Recognize microbial structures Acquired (adaptive) immunity (specific) Lymphocytes, B cells, antibodies Second line of defense, it is invoked if innate immunity is not enough Stimulated by exposure to infectious agents and adapts to the infection à respond more vigorously to repeated exposures (e.g. vaccinations for specific diseases)

Immune cells (blood) Blood cells Red blood cells (erythrocytes) White blood cells (leukocytes) (1%) Platelets Blood analysis shows higher % of leukocytes à if leukocytes % ñ, sign that immune system is activated at someplace in the body (we don t know where!) Blood transports immune cells from one tissue to another. Important immune actions take place in tissues.

Leukocytes GRANULOCYTES (innate immune response) Dominate the first hours of inflammatory process; Not activated: in blood. Activated: invade the infection site, and more are produced. Brief life (6-36h in blood and 2-5 days in tissues). Types: - - - Eosinophil Basophil Neutrophil

Leukocytes GRANULOCYTES (innate immune response) Dominate the first hours of inflammatory process; Not activated: in blood. Activated: invade the infection site, and more are produced. MONOCYTES (innate immune response) Not activated: in blood. Activated: invade the infection site. Brief life (6-36h) Brief life (6-36h in blood and 2-5 days in tissues). Types: - - - Eosinophil Basophil Neutrophil In the site of infection they differentiate into: - Macrophages (innate I.R.) - Dendritic cells (acquired I.R.)

Leukocytes LYMPHOCYTES/PLASMA CELLS (acquired immune response) Part of acquired immure response. Types: - T and B cells - Natural killer cells Memory cells: Responsible for a more rapid response if one is exposed to the same pathogen for second time. Effector cells: Produce antibodies or take other action to remove foreign invaders (specific).

Leukocytes LYMPHOCYTES/PLASMA CELLS (acquired immune response) Part of acquired immure response. MEGAKARYOCYTES (innate immune response) Found in the bone marrow. Types: - T and B cells - Natural killer cells Function: It fragments to form platelets (that aid in blood cloeing). Memory cells: Responsible for a more rapid response if one is exposed to the same pathogen for second time. Effector cells: Produce antibodies or take other action to remove foreign invaders (specific).

Neutrophils: functions Dominate the first hours of inflammatory process (brief life). Not activated: in blood. Activated: invade the infection site, and more are produced. Neutrophils perform several actions with the aim to destroy the foreign pathogens: Phagocytosis Respiratory burst Secretion of chemical mediators

Neutrophils: functions Phagocytosis: ability to engulf, destroy and eliminate microorganisms, damaged cells, or foreign materials.

Neutrophils: functions Respiratory burst: Release of reactive oxygen and nitrogen species (radicals and strong oxidizers). à Kill foreign organisms à Promote corrosion and oxidative degradation of biomaterials It also causes unwanted tissue damage (keep it localized!).

Neutrophils: functions Secretion of chemical mediators, e.g. cytokines and chemokines. Proteins that orchestrate several cell actions: - - - - - - - Proliferation Differentiation Recruitment Maturation Communication between cells Cell death Regulate inflammatory reactions (more information later)

Monocytes/macrophages Due to the signals released by the neutrophils, monocytes arrive at the injury site. Monocytes (blood) Monocytes (tissue) Differentiate Cells enlarge and form large quantity of lysosomes* Macrophages (tissue) Over days, dominant cell type *Lysosomes: Vesicle in the cell cytoplasm that contains hydrolytic enzymes

Macrophages: functions Similar functions to that of the neutrophil, but with greater killing capacities. Phagocytosis Secretion of chemical mediators Role as antigen- presenting cells

Macrophages: functions Role as antigen- presenting cells Macrophages phagocyte a microbe, make pieces of it à macrophage displays the antigen (small protein that acts as a fingerprint) on its membrane to call lymphocytes to aeack the invading microorganism. Activation of the acquired immune response is triggered.

2. Inflammation caused by the implantation of a biomaterial

Innate immunity Combination of defenses to reduce the number of invading organisms: 1. Anatomic barriers (skin and mucous membranes) 2. Physiologic barriers (temperature of body, low ph in stomach) 3. Phagocytic cells (granulocytes and macrophages) 4. Acute inflammation Microorganisms Body

Defense mechanisms after implantation Microorganisms Body Defense mechanisms (originally intended to protect against pathogens)

Defense mechanisms after implantation Microorganisms Biomaterial Body Defense mechanisms (originally intended to protect against pathogens) Most biomaterials cause acute inflammation

Defense mechanisms after implantation The implantation of a biomaterial results in disruption of tissues, causing perturbation of homeostasis* Innate immune response will try to eliminate the biomaterial in an aeempt to return to homeostasis ACUTE INFLAMMATION (< 48h) No successful (biomaterial remains) Acquired immune response and innate immune response work together CHRONIC INFLAMMATION (days- months) Both innate and acquired immune response are highly intertwined, sharing signaling molecules *Homeostasis: variables of the body are regulated so that internal conditions remain stable and relatively constant.

Inflammatory process Inflammation process after the implantation of a biomaterial inflammation Implantation of a biomaterial = perturbation of body s homeostasis The body aeemps to restore the tissue to its previous state Resolution: restitution of the pre- existing homeostasis Accute inflammation Chronic inflammation Immediate response to tissue injury (< 48h) (neutrophils, monocytes) - Immune response - Repair phase (weeks, months) (macrophages, FBGCs, lymphocytes)

Inflammation caused by a biomaterial Inflammation aims to: 1) Contain or neutralize the foreign agent; and 2) Heal the implant site: replace the injured tissue by new tissue The interaction between immune cells and the surface of a material is believed to determine the eventual success or failure of an implanted device.

3. Acute inflammation

Acute inflammation 1. Vasodilatation (expand) of blood vessels in nearby regions à redness and heating of tissue 2. Permeability of blood vessels increase, causing leakage of fluid into interstitial (surrounding) space (can cause swelling of tissues) 3. Blood proteins (i.e. fibrinogen) will clot the vessel (anatomical barrier to invading organisms). Cloeing cascade release kinins that cause pain. 4. Granulocytes and monocytes are aeracted to the injured tissue and they may call for more cellsà phagocytose debris and foreign organisms 5. Monocytes differentiate into macrophages in the injured tissue

Clinical signs of acute inflammation 1. Rubor (redness) 2. Tumor (swelling = leakage of interstitial fluid) 3. Calore (tissue heating) 4. Dolore (pain): due to release of kinins 5. Loss of function vasodilatation of blood vessels

Acute inflammation: neutrophils migration Granulocytes Endothelial cells or ECM molecules Capture/ Anchor Rolling Activation Adhesion Diapedesis Transversing basal lamina Muller. Lab Invest 82 (2002) 521 Migration through ECM Basal lamina Granulocytes (neutrophils) and monocytes: blood vessels extravasation tissue Extravasation in brief: Bind to vascular endothelium à penetrate the endothelial layer lining the blood vessel à migrate to the area of inflammation

Acute inflammation: neutrophils migration Capture/ Anchor Rolling Granulocytes Activation Adhesion Diapedesis Transversing basal lamina Endothelial cells or ECM molecules Muller. Lab Invest 82 (2002) 521 Migration through ECM Basal lamina 1. Rolling: Neutrophils are carried along by the blood, they bind briefly to the vascular endothelium through low- affinity selectin- carbohydrate interactions à cells anchored briefly to the endothelium and then the flowing blood dislodges them. This is repeated over and over, appearance that cells are rolling along the surface of the blood vessels.

Acute inflammation: neutrophils migration Capture/ Anchor Rolling Granulocytes Activation Adhesion Diapedesis Transversing basal lamina Endothelial cells or ECM molecules Muller. Lab Invest 82 (2002) 521 Migration through ECM Basal lamina 2. Activation: Rolling neutrophils are activated by chemoaxractants (substances that cause migration towards the damaged tissue; movement called chemotaxis). The two most prominent activating chemoaxractants are: - - Interleukin- 8 (IL- 8) Macrophage inflammatory protein- 1β (MIP- 1β) IL- 8 and MIP- 1β bind to specific receptors on the neutrophil s surface and induce induce a conformational change in integrin molecules in the cell membrane. The integrins now have a higher affinity for the immunoglobulin superfamily (cell adhesion molecules, CAM) found on the surface of the endothelium.

Acute inflammation: neutrophils migration Capture/ Anchor Rolling Granulocytes Activation Adhesion Diapedesis Transversing basal lamina Endothelial cells or ECM molecules Muller. Lab Invest 82 (2002) 521 Migration through ECM Basal lamina 3. Adhesion: The strong interaction between integrin receptors on the neutrophil and CAMs presented by endothelial cells leads to a firm adhesion to the endothelium. 4. Transendothelial migration: Neutrophil goes through the endothelial membrane and reached the inflamed tissue. The cell accomplishes this through diapedesis, i.e. squeezing parts of the cell at a time through small spaces between endothelial cells. Once in the target tissue, neutrophils express high levels of receptors for further chemoaeractants, since more neutrophils are needed at the area.

Cytokines involved (a few of them) Interleukin- 1 (IL- 1): Promotes granulocyte migration, activates lymphocytes, increases production of IL- 8 IL- 8: Aeracts neutrophils, promotes neutrophil integrin- CAM interaction Macrophage inflammatory protein 1α (MIP- 1α): Aeracts monocytes Tumor necrosis factor- α (TNF- α): Promotes granulocyte migration, activates lymphocytes, increases production of IL- 8 Transforming growth factor- β (TGF- β): Limits inflammatory response Cytokines produced by innate immune response acts on: IL- 1, IL- 8, MIP- 1α, MIP- 1β, TGF- β, TNF- α à Monocytes, granulocytes (innate I.R.) IL- 1, IL- 6, TNF- α à Lymphocytes (acquired I.R.) Granulocytes: eosinophils, basophils, neutrophils CAM = cell adhesion molecules

Types of cytokines Cytokines are classified depending on their ability to stimulate inflammation and wound healing. Secretion is brief and local Pro- inflammatory/anti- wound healing Anti- inflammatory/pro- wound healing Pro- inflammatory/pro- wound healing Anti- inflammatory/anti- wound healing

Termination of acute inflammation Acute inflammation is a double- edged sword The acute inflammatory response is well designed to help in recovering homeostasis and preventing the invasion of foreign organisms after the injury. However, reactions occurring during acute inflammation can damage the tissue if they are widespread or continued for a long time! Ways to avoid widespread and tissue damage due to inflammation: The chemical mediators involved in inflammation are quickly inactivated or destroyed in solution à their action is local. During inflammation, the interaction of a variety of chemical mediators provides a system of checks and balances to ensure that the effects are localized.

Reducing/ending acute inflammation The body has means to reduce or end acute inflammation. 1) Cleavage of chemokines, down- regulation inflammatory mediators, apoptosis* immune cells. Example Release of anti- inflammatory cytokine, e.g. transforming growth factor- β (TGF- β). TGF- β inhibits the activation of certain inflammatory cells. Apoptosis = programmed cell death

Means to reduce/end acute inflammation 2) Release of receptor agonists. The same cells that produce IL- 1, produce also IL- 1 receptor agonist (IL- 1ra), molecule with a similar chemical structure to IL- 1 (binds to the same receptors) but does NOT stimulate them. This is a way, target cell s activation is inhibited by binding IL- 1ra. Soluble- IL- 1 Soluble- IL- 1ra IL- 1 receptor Signal No signal Cell surface

4. Wound healing in the presence of biomaterials

Paths after acute inflammation After acute inflammation Chronic inflammation Foreign body reaction Resolution J Persistence of chronic inflammation L Granulation tissue Fibrous encapsulation (normal wound- healing response for a non- degradable biomaterial)

Chronic inflammation Characterized by the presence of mononuclear cells (lymphocytes and plasma B cells), indicating that the material has triggered an acquired immune response. Chronic inflammation can be a caused by: - Chemical or physical properties of the biomaterial - - Topography Shape - Motion at the implant site Plasma B cells: leukocytes that secrete large amounts of antibodies (transported in blood)

Foreign body reaction Large biomaterials cause the formation of foreign body giant cells (FBGC) è macrophages coalesce to FBGCs in an aeempt to phagocytize the biomaterial. Macrophages FBGC Large multi- nucleated cell Layer of FBGCs surrounding a non- phagocytosable particle are called granulomas. FBGC = Foreign body giant cells

Formation of granulation tissue Implantation of a biomaterial After 1 day: FBGCs* release pro- fibrotic factors (TGF- β and PDGF*) that trigger action of fibroblasts and endothelial cells. After 3-5 days: Granulation tissue is formed. This tissue looks granular due to the creation of many vascular buds growing from existing blood vessels (neovascularization or angiogenesis). Fibroblasts proliferate and also take on features of smooth muscle cells (myofibroblasts). Myofibroblasts are responsible for wound contraction, which results in faster healing due to a decrease in the overall defect size. *FBGC = Foreign body giant cells *PDGF = platelet derived growth factor *Fibroblasts: cell commired to synthesize connective tissues by producing ECM (collagen + proteoglycans)

Formation of granulation tissue Granulation tissue Cellular debris Fibrinoid necrosis Granulation tissue

Fibrous encapsulation This is the final stage for non- degradable implants. It involves maturation of granulation tissue (large blood vessels and alignment of collagen fibers in response to local mechanical forces) à Biomaterial isolated from the body. Fibrous encapsulation is considered an acceptable result after implantation of many biomaterials

Quiz 1 Scanning electron microscopy images of a Polyurethane surface from an in vivo cage study showing the morphological progression of the foreign body reaction. - - Can you guess the sequence of events? What is happening in each stage? Anderson et al. Semin Immunol. 2008; 20(2): 86 100.

Quiz 1: answer Scanning electron microscopy images of a Polyurethane surface from an in vivo cage study showing the morphological progression of the foreign body reaction. Sequence of events: (A) monocyte adhesion (0 days) (B) monocyte- to- macrophage development (3 days) (C) ongoing macrophage- macrophage fusion (7 days) (D) foreign body giant cells (14 days). Anderson et al. Semin Immunol. 2008; 20(2): 86 100.

Chronic inflammation Persistence of chronic inflammation is considered pathologic (implant may have to be removed)

Resolution of inflammation Overall goal of the inflammation response and wound healing is to restore the homeostasis to the body. The formation of an new equilibrium state can be considered a resolution of this response. There are 4 main types of possible resolutions: Extrusion: If implant is in contact with epithelial tissue (top layer of skin), the material can be forced out of the body. Resorption: In the case of a biodegradable material (depending on the degradation rate, fibrous capsule will be formed or not. Integration: Close approximation of host tissue to the implant, with no intervening fibrous capsule (e.g. titanium in bone). C Encapsulation: Formation of a fibrous capsule around the tissue (acceptable for biomaterial implantation, not for tissue- engineered* product) *Tissue engineering: Field in which the overriding goal is to create a fully functional tissue

Quiz 2: Which will be the outcome? Nanomaterial 10-200 nm If degradable (A) If non- degradable (B) Large biomaterial (mm cm) Non- degradable (C) 20 µμm Inflammatory cell, e.g. macrophage

Quiz 2: Which will be the outcome? Nanomaterial 10-200 nm If degradable If non- degradable Phagocyted and eliminated Phagocyted but not eliminated. It may elicit further inflammatory response of cells, may damage surrounding tissue. Frustrated phagocytoses due to size disparity à FBGCs à fibrous encapsulation Large biomaterial (mm cm) Non- degradable In some cases (e.g. pure Ti), integration may occur Extrusion (when in contact with the skin)

5. Tailoring immune responses of biomaterials

New trends in biomaterials The immune response is crucial for tissue repair. Aim: Design materials to elicit an appropriate immune response at implantation site ( immuno- modulating capacity) FBGC = foreign body giant cell

Biomaterial properties Some properties of the biomaterials will affect the inflammation. Biomaterials properties Size Shape Topography Weeability Charges Intensity and duration of the inflammation Biocompatibility* of the material *Good biocompatibility: biomaterial performs as intended and presents no significant harm to the patient.

Passive modulation Modification of superficial properties to guide inflammatory response: - Size - Shape Tailoring immune response - Topography - Weeability - Charges Strategies: Active modulation (molecules designed to target cell behaviour): Incorporation of bioactive molecules: - - - Functionalization of biomaterial surfaces to direct responses of inflammatory cells. Coupling of anti- inflammatory drugs to biomaterials. Delivery of growth factors: to control adhesion, migration, proliferation, and differentiation of fibroblasts and endothelial cells in wound healing.

6. Research studies

1) Surface biomaterial Hydroxyapatite materials prepared from a calcium phosphate cement Plate- like crystals (C- HA) Needle- like crystals (F- HA) Mestres et al. Plos One (2015, accepted)

1) Surface biomaterial Macrophages on material surface Mestres et al. Plos One (2015, accepted)

1) Surface biomaterial Release of ROS caused by the activation of macrophages. Significantly higher on C- HA. TCPS = tissue cultured polystyrene Same cell number in both materials Mestres et al. Plos One (2015, accepted)

2) Particle size Unmodified amorphous silica particles with diameters of 30, 70, 300, or 1 000 nm (designated nsp30, nsp70, nsp300, and msp1000, respectively) Size distribution of silica particles (measured by a dynamic light- scaeering). Morishige et al. Arch Toxicol (2012) 86:1297 1307

2) Particle size Macrophages were treated with nsp70 particles for 4 h. Cells were observed by TEM. Morishige et al. Arch Toxicol (2012) 86:1297 1307

2) Particle size TNF- α production levels in vitro. Macrophages were treated with each silica particle or no particles (Non) for 12 h, and then, TNF- α production levels in the culture supernatants were measured. Inflammatory response is particle- size dependent. Nanosilica particles induced stronger inflammatory responses (higher TNF- α production) than microsilica particles. Morishige et al. Arch Toxicol (2012) 86:1297 1307

2) Particle size Amorphous silica particles were used unmodified (nsp70) and modified with surface carboxyl groups (nsp70- C). Non = without particles SP70- C suppressed TNF- α production Morishige et al. Arch Toxicol (2012) 86:1297 1307

3) Imaging inflammation in vivo Near- infrared fluorescence imaging of ROS as a surrogate measure for monitoring implant- associated inflammation in vivo. Reaction with superoxide or hydroxyl radical oxidizes the hydrocyanines into fluorescent cyanine dyes, which can be detected in vivo by fluorescent imaging. Selvam et al. Biomaterials 32 (2011) 7785 7792

3) Imaging inflammation in vivo Hydrocyanine ROS sensors delivered either locally or intravenously in living mice. ROS released by inflammatory cells in response to implanted biomaterials. Aeenuation of inflammatory responses in response to controlled release of anti- inflammatory agents Selvam et al. Biomaterials 32 (2011) 7785 7792

3) Imaging inflammation in vivo Mice undergoing the same surgical procedure but receiving no biomaterial implants were used as sham controls to account for surgery- associated trauma/inflammation Selvam et al. Biomaterials 32 (2011) 7785 7792

Summary

Type of cells involved Granulocytes (neutrophils) (< 24 h) ê Monocytes ê Acute inflammatory response Monocytes emigrate to the damaged tissue and differentiate into macrophages. Neutrophils Macrophages Neovascularization Foreign body giant cells Fibroblasts Fibrosis Mononuclear leukocytes (monocytes) Chronic inflammatory response

Anderson et al. Semin Immunol. 2008 April ; 20(2): 86 100. Cells differentiation NEUTROPHIL MONOCYTE MACROPHAGE FOREIGN BODY GIANT CELL Blood Tissue Tissue/Biomaterial Biomaterial ACUTE INFLAMMATION CHRONIC INFLAMMATION

Summary Implant may fail Persistence of chronic inflammation FBGCs formation Implantation of biomaterial = INJURY Accute inflammation Inflammatory cells: - Aeracted by injured site - Release of chemical mediators (e.g. cytokines) and phagocytosis Chronic inflammation & Foreign body reaction Resolution Non- degradable material Encapsulation Degradable material Integration Successful tissue repair

For further questions / comments, feel free to e- mail me: gemma.mestres@angstrom.uu.se