1 3.051J/20.340J Lecture 8: Cell-Surface Interactions: Host Responses to Biomaterials (Part II) Implantation of a biomaterial initiates the inflammatory response: - response of vascularized tissue to local injury - severity indicates biocompatibility of material Cooperative Signaling Cascades: 1. Coagulation Cascade initiated by adsorbed2. Complement Alternative Pathway proteins The complement is a component of the immune system. Immune system function: to protect against pathogens Innate (Native) Immunity Adaptive (Aquired) Immunity - first line of defense - specificity to distinct foreign biomolecules (antigens) - nonspecific response to invading pathogens - memory of exposure - elicits adaptive response Physical/chemical barriers: Blood proteins: epithelia, antimicrobial proteins antibodies (immunoglobulins), cytokines Blood proteins: complement; cytokines (regulatory) Cells: lymphocytes (T cells, B cells) Cells: phagocytes (macrophages, neutrophils), natural killer cells2 3.051J/20.340J Complement - system of >30 proteins that mediate immune response - discriminates “foreign” from “self” through adsorbed proteins/ protein fragments (C3b, C4b) - recruit and activate phagocytes (C3a, C5a) - lysis of pathogens via membrane pore formation (C5b, C6-C9) 3 recognized pathways (to C5 convertase) Classical pathway: antigen-antibody immune complex (IC) binds and activates C1 (autocatalytic proteolysis) initiating an enzymatic cascade C1 → C1s C4 → C4b C2 → C2b C3 → C3b C5 → C5a/C5b soluble fragment (16 kDa): insoluble fragment (170 kDa): recruits phagocytes by chemotaxis initiates membrane attack complex (MAC) C5b•C6•C7•C8•C9 MAC pore formation compromises bacterial cell membrane3 3.051J/20.340J Lectin pathway (discovered in 1990’s): mannan binding lectin (MBL) bacteria cell wall binds carbohydrates on pathogen (gram negative) MBL-associated serine proteases (MASP-1, -2) complexes with MBL activated MASP’s cleave C4 → C4b remaining cascade follows classical pathway Alternative pathway: nonselective pathway of complement (any foreign surface) C3 → C3b occurs continuously in plasma at low frequency C3b adsorbs on foreign surfaces (biomaterial) cofactor B → C3b•Bb complex amplifies C3 → C3b C3b•C3b•Bb complex C5 → C5a/C5b Soluble complement protein fragments C3a and C5a recruit phagocytes to site of injury4 3.051J/20.340J Inflammatory Response to Implanted Biomaterials INJURY TIME Seconds/Minutes Blood-Materials Interactions Hours Provisional Matrix Formation Acute Inflammation Days Chronic Inflammation Granulation Tissue Foreign Body Reaction Weeks Fibrosis/Fibrous Capsule Formation Cell fibroblasts macrophages foreign body giant cells Activity neutrophils hrs days weeks time5 3.051J/20.340J • Endothelial cellsenzymes (1.3 mg/ml) changes conformation ⇒ cleavedC3 lining capillaries near injured site release plasma protein C3 into fragments C3b fragment C3a fragment C3b attaches to biomtl or injurious agent C3a diffuses into medium ⇒ soluble surface ⇒ insoluble ligand for leukocyte ligand for leukocyte receptors receptors IMMUNE CELLC3b catalyzes C5 cleavage to C5a ⇒ RECRUITMENTsoluble ligand for leukocyte receptors6 3.051J/20.340J Neutrophils (PMN’s, polymorphonuclear leukocytes) Associated with acute inflammatory response (minutes→1-2 days) ¾ “first responders” 3-5M/ml (short-lived) ¾ bind C3a/C5a via complement receptors (CR’s) ¾ become hyperadherent by ↑ CR3 (integrin CD11b/CD18) surface expression – attach to vasculature via endothelial ICAMs ¾ chemotactic to C5a: migrate to inflammation site On site, neutrophils bind to C3b, catalyzing release of cytotoxic species: H2O2, O2 −• (superoxide radical), OH•, enzymes ⇒ attack/engulf/degrade invading microbes Released products from neutrophils, activated platelets and endothelial cells, along with fibrin, form the provisional matrix - scaffold for cell attachment - sustained release of signaling molecules7 3.051J/20.340J Monocytes (0.2-0.6M/ml) ¾ bind C3a/C5a ⇒ follow the course of neutrophils ¾ Evolve to macrophages ¾ Associated with chronic inflammation days→ weeks/months (or even a lifetime) On site, macrophages bind C3b, secrete reactive species, enzymes, cytokines (immune cell regulators, ex. IL-1), fibronectin, growth factors (ex. fibroblast growth factor, epidermal growth factor), coagulation factors Macrophage response depends on foreign material properties… ¾ fluids or small particles (micron-sized) → engulfed & degraded “phagocytosis” with adsorbed ligands fusion of phagocytic foreign material vesicles and lysosomes Degraded biomolecule products: lysosome amino acids, sugars, lipids Nondegradable products accumulate8 3.051J/20.340J ¾ Numerous particulate debris or materials with high roughness → fusion of macrophages into multinuclear foreign body giant cells (FBGCs) ¾ smooth, inert implants FBGCs absent (nothing to engulf) → macrophage layer surrounds implant Macrophage/FBGC products (FN, FGF) recruit fibroblasts Fibroblasts (connective tissue cells) ¾ deposit collagen → pink “granulation tissue” (appears in 3-5 days) ¾ accompanied by capillary sprouting (angiogenesis) Wound healing histology: foreign body reaction presence of FBGCs/macrophages, granulation tissue, capillaries at tissue/material interface ¾ Connective tissue remodeling ⇒ thin, encapsulating fibrous layer (fibrosis) isolates implant and foreign body rxn (weeks)3.051J/20.340J 9 Formation of scar tissue vs. parenchymal tissue (tissue of specialized function) depends on: ¾ extent of parenchymal tissue damage (esp. tissue framework) ¾ parenchymal cell proliferation capacity Cell Regeneration Capability Category Normal replic. rate Response to injury Examples renewing/ labile High; via stem cell differentiation modest ↑ skin, intenstinal mucosa, bone marrow Expanding/ stable Low large ↑ endothelium, fibroblasts, hepatocytes, osteoblasts Static/ permanent None No replication heart muscle cells, nerve cells FBGC formed at implant site. Arrows point to nuclei. (J.S. Belkas et al., Biomaterials 26 (2005) 1741.) Fibrous capsule formation around porous P(HEMA-co-MMA) nerve conduit (J.S. Belkas et al., Biomaterials 26 (2005) 1741.) Photos removed for copyright reasons.10 3.051J/20.340J Implant biocompatibility is assessed largely by