Kim, Hong Sung, Ph.D. BMS. NRLS. PNU 2016

Interactions between Biomaterials and Tissue

Biomaterials II

Introduction

• Part 1 – Biomaterial Science (생체재료학 1) • Part 2 – Biological-Medical Science (생체재료학 2)

• Focus in materials - cells(tissue or blood) interactions – Concept: Environment, Response, Interactions – Host Reactions: injury, immunity, disease, healing – Degradation: products, effects, calcification

• 교재: Biomaterials Science, An Introduction to Materials in Medicine, 2nd Edition, Edited by Buddy D. Ratner, et al., Elsevier Academic Press

• 그림과 도표는 교재에 있는 것을 참조

Chapter 1 The Interactions between Biomaterials and Tissues 1.1 Background Concept 1.2 The Role of Adsorbed Proteins

The Effects of Adhesion Proteins, Depletion, Inhibition of Receptor Activity with Antibody, Adsorption Behavior of Proteins at Solid/Liquid Interfaces, Monolayer Model, Competitive Adsorption, Molecular spreading, Biological Changes, Time-dependent transition, Importance of Adsorbed Proteins

1.3 Cells and Cell injury Normal Cell, Plasma Membrane, Passage through plasma membrane, Unique transporter, Osmosis, Endocytosis, Receptor-meditated endocytosis, Endosome, Transcytosis & Exocytosis, Cell Communication, Extracellular signaling, Intracellular & cell-surface Receptors, Cytoskeleton, Actin, Intermediate Filaments, Microtubules, Cell-Cell connections, Desmosomes, Nucleus, Chromatin, Nuclear Envelope, Nuclear pores, Biosynthetic Machinery, Smooth endoplasmic reticulum, Waste Disposal, Materials destined for catabolism arrive in lysosomes, Proteasomes, Energy Generation, Mitochondria Structure, Cell Specialization and Differentiation, Cell Injury & Regeneration, The Principal Adaptive Response, Injury, Cancer, Causes, Pathogenesis, Responses, Cell Response in Hypoxia, Necrosis, Apoptosis

1.4 Tissues, ECM, Cell-Biomaterial Interactions Need for Tissue Perfusion, Extracellular matrix, Collagens and elastin, Amorphous Matrix, Adaptor/Adhesive Molecules, Cell-Matrix Interactions, Basic Tissues, Organs, TISSUE RESPONSE TO INJURY(Inflammation and Repair, Cell Regenerative Capacity, Extracellular Matrix Remodeling), CELL/TISSUE-BIOMATERIALS INTERACTIONS, TECHNIQUES FOR ANALYSIS OF CELLS AND TISSUES(LIGHT Microscopy, Tissue Sample, Fixation, Dehydration and Embedding, Sectioning, Staining, Special Staining, Electron Microscopy, Three-Dimensional Interpretation, Artifacts, Identification)

1.5 Mechanical Forces on Cells VASCULAR CELL RESPONES, SKELETAL CELL RESPONSES

Index

Chapter 2 Host Reactions to Biomaterials 2.1 Introduction

THE INFLAMMATORY REACTION TO BIOMATERIALS, SYSTEMIC AND REMOTE EFFECTS, THROMBOEMBOLIC COMPLICATIONS, TUMORIGENESIS, INFECTION

2.2 Inflammation, Wound healing, Foreign body response OVERVIEW, ACUTE INFLAMMATION, CHRONIC INFLAMMATION, GRANULATION TISSUE, FOREIGN-BODY REACTION, FIBROSIS/FIBROUS ENCAPSULATION

2.3 Systemic Toxicity and Hypersensitivity KINETICS AND NATURE OF BIOMATERIALS COMPONENTS, TOXICODYNAMIC CONSIDERATIONS, ADVERSE EFFECTS OF DEFENSE MECHANISMS, HYPERSENSITIVITY AND IMMUNOTOXICITY, ALLERGY AND BIOMATERIALS, TYPES OF ALLERGIES( Type I Hypersensitivity, Type IV Hypersensitivity), ATOPY, IMMUNOLOGIC TOXICITY OF MEDICAL DEVICES, OTHER INTERACTIONS, CONCLUDING REMARKS

2.4 Tumorigenesis and Biomaterials GENERAL CONCEPTS, ASSOCIATION OF IMPLANTS WITH HUMAN AND ANIMAL TUMORS, PATHOBIOLOGY OF FOREIGN BODY TUMORIGENESIS, CONCLUSIONS

2.5 Biofilms, Biomaterials, and Device-Related Infections BIOFILM MICROBIOLOGY, Bacterial Adhesion to Surfaces, Biofilm Formation on Surfaces, Natural Control of Biofilm Formation on Surfaces, Novel Engineering Approaches to Biofilm Control, BIOFILM-RESISTANT BIOMATERIALS,

Index

Chapter 3 Degradation of Materials in the Biological Environment 3.1 Introduction 3.2 Chemical and Biochemical Degradation of Polymers

POLYMER DEGRANDATION PROCESSES, HYDROLYTIC BIODEGRANDATION, OXIDATIVE BIODEGRADATION

3.3 Degradative Effects of the Biological Environment on Metals and Ceramics

METALLIC CORROSION, INFLUENCE OF THE BIOLOGICAL ENVIRONMENT, CORROSION AND CORROSION CONTROL IN THE BIOLOGICAL ENVIRONMENT, CERAMIC DEGRANDATION

3.4 Pathological Calcification of Biomaterials The spectrum of pathologic biomaterials and medical device calcification, ASSESSMENT OF BIOMATERIALS CALCIFICATION, PATHOPHYSIOLOGY, PREVENTION OF CALCIFICATION

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CHAPTER

1 The Interactions between Biomaterials and Tissues

1.1 Background Concept

• After implantation: Proteins absorb (< 1 sec.), and monolayer (to minutes) on biomaterial surface.

• Cells see primarily a protein layer, and respond to proteins. (Ch.1.2)

• In response to proteins on surface, cells adhere, release active compound, recruit other cells, grow in size, replicate, and die.

• Cells secrete ECM molecules, angiogenesis, and vasculogenesis for new tissue with nutrition/remove wasters. (Ch.1.3)

• Development & response to injury of tissues must be understood to appreciate the interplay between materials and tissues (Ch. 1.4)

• Cells & tissues respond to mechanical forces. Triangle shape show more fibrous reaction than circle. Blood cell & Endothelial cell distinctly different behavior depending upon shear forces. (Ch. 1.5)

1.2 The Role of Absorbed Proteins

• The replacement of injured tissues with materials not of biologic origin is the central approach.

• It is due to not attacked by the immune system. • But including the clotting of blood and foreign body

reaction. • This reaction is adsorption of adhesion proteins to

surface of materials recognized by integrin receptors present on most cells.

• The interaction of the adhesion proteins with the receptors is a major cellular recognition system for biomaterials. (The aim of this chapter.)

• Integrin receptors of cell mediates the biological activity with the adsorbed proteins on foreign body.

• The receptors recognized and adhere only to one protein (circle type).

• Solution and solid phase of the same protein have different biological activity.

The Effects of Adhesion Proteins • Pre-adsorption with purified adhesion proteins

– Fibronectin pre-adsorption greatly increases adhesion of fibroblasts

– Whereas albumin preadsorption prevents it • Adhesion proteins promote of the cell onto the surface.

– Spreading is only 5% in the absence of fibronectin. – But increases nearly 100% as fibronectin conc. in

preadsorption solu. are increased from 0.03 to 3ug/ml.

Hydroph□□ic

Fibronectin & Vitronectin Fibronectin (섬유결합소, 접착성당단백 )

• Fibroblast & endothelial cell 에서 합성

• Proteoglycan & cell 결합 영역 존재

• 2개의 peptide 사슬이 disulfide 결합된 dimer:

• 말단-결합부위 :

• Amino end group - adhesion proteins 순 NH2-Fibrin-collagen-DNA-heparin-cell-fibrin--

NH2-heparin- - - - - - - - - - - - - - - - - - - - - - -ss-

• 440 KDa (1250개 amino acids)

• 세포막 integrin 인식: 세포와 ECM 연결체 • 세포유착, 이동, 상처치유 관여 • 암세포 전이: 세포표면에 fibronectin양 감소

Vitronectin • 혈청에 포함, 75 KDa • N-terminal Somatomedin B –

hematopexin – C-terminal • 염증 시 치유 유도 역할 • RGD 서열 존재 • Collagen, Heparin, Thrombin

등과 결합 Adhesive amino acid sequence

• ARG-GLY-ASP (RGD) • ARG-GLY-ASP-SER (RGDS) • LEU-ASP-VAL (LDV) • ARG-GLU-ASP-VAL (REDV)

Depletion • Implants are exposed to complex mixture of proteins;

plasma or serum • Very little of the adhesion protein may absorb to the

limited surface sites, outcompeted by other proteins. • To understand the role of an adhesion protein is to study

the effect of selective depletion from the complex mixture.

• Vitronectin as opposed to fibronectin in mediating attachment and spreading of cells on many surfaces.

• On TCPS, fibronectin removal has little effect, whereas vitronectin removal greatly reduces adhesion of endothelial cells.

• On surface modified by ammonia in glow discharge, fibronectin removal greatly reduces adhesion, whereas vitronectin removal has little effect.

• The results on TCPS are more typical. • Why did the reversed effect appear? • Adsorbed fibrinogen plays in adhesion of platelets,

neutrophils, and macrophages. • Platelet adhesion to surface preadsorbed with plasma

deficient in fibrinogen is much less than that with normal plasma.

Why declines ?

• When mice are depleted of fibrinogen, the adhesion of neutrophils and macrophages to a polymer implanted in peritoneal cavity is greatly reduced.

• Previously, it had been thought that either complement or IgG would be the main adhesion proteins for neutrophils and monocytes.

• It appears that an integrin receptor for fibrinogen (CD11b/CD18, known as Mac-1) plays a major role, at least during the initial or acute phase of the foreign body response.

Inhibition of Receptor Activity with Antibody • Another way to show the role of adhesion proteins in

cell interaction with biomaterial is to add specific inhibitors; to add an antibody that binds to the integrin receptor, blocking access to the adhesion protein.

• Platelet deposition is largely inhibited by antibodies to the glycoprotein IIb/IIIa receptor in in-vivo with a vascular grafts.

• Platelet deposition is inhibited by anti-glycoprotein IIb/IIIa in in vitro polyurethane Biomer. (Ch.3.2: Fig.4)

Adsorption Behavior of Proteins at Solid/Liquid Interfaces • Adsorption transforms the interface

– Surface of polystyrene exposed to a protein solu. for a short time.

– The surface become coated with a layer of hydrophilic protein adsorbate.

• Rapid Adsorption Kinetics and Irreversibility – Very rapid initial phase, followed by a slower

phase. (Ch.3.2: Fig.6)

Monolayer Model • Adsorption isotherm of saturation effect (Ch.3.2:Fig.7) • Steady state adsorption (2 hours or more) • Typically reaches a plateau or saturation value for

close-packed monolayer of protein ( 0.1 to 0.5 ug/cm2) • This amount is adsorption of three main proteins in

plasma (albumin, IgG, and fibrinogen) • When monolayer is the limit, Langmuir isotherm • Adsorption rises much more, Freundlich isotherm

• Distribution ( I ) : A = kX (분배형)

• Langmuir ( II ) : A = X/(X-1) (단층형)

• Freundlich ( III ) : A = Xb (다층형)

• BET ( IV ) : s curve (복합형)

• Dual sorption ( V ) : (이원수착)

흡착기구 ( Type )

Competitive Adsorption • Adsorption is selective, leading to enrichment in certain

proteins. • When a radio-labeled protein(fibrinogen, A) is mixed

albumin(B), amount of competing protein B needed to inhibit the adsorption of A is different in each surface.

• On PE, adsorption of fibrinogen requires 10-fold excess by competing protein albumin,

• but inhibited 10% of higher affinity protein hemoglobin. (Ch.3.2: Fig. 8)

• Although fibrinogen is only 3rd most concentrated protein in plasma, after IgG and albumin, biomaterials exposed to plasma are enriched in fibrinogen.

혈액 중 hemoglobin 농도는 14,000 mg/dl 정도이다. 그럼에도 implant 표면에 fibrinogen이 많이 붙는 이유는?

• Albumin, lower-affinity protein, has much higher conc. than fibrinogen in plasma(400 mg/dl). But its surface conc. absorbed from plasma is typically about the same as fibrinogen.

• Adsorption amount is affected by its concentration, competitiveness, and its affinity of the surface.

• Hemoglobin is present in very low conc. in plasma (1 mg/dl or less), but it is still absorbed in amounts similar to the more predominant proteins because of its high surface affinity.

Albumin: 4000 mg/dl, Fibrinogen: 400 Globulin: 1700 Glucose: 100 ImmunoG: 1600 (17%)

Effects for competitive adsorption

Molecular spreading • Proteins that adsorbed to solid surfaces can undergo

conformational changes because of low structural stability and further bond formation with the surface.

• Physicochemical study: – Less stable proteins are more adsorptive. – With DSC, transition enthalpy of lysozyme adsorbed

to – charged PS (0-170 kJ/mol) was much less than for in solution (600 kJ/mol for native protein).

– it has already unfolded upon adsorption.

Synthesis of Human Lysozyme

Proc. Natl. Acad. Sci. U.S.A. 104 (12): 4846–51 (2007) Durek T, Torbeev VY, Kent SB

– Pyrolytic carbon induces complete unfolding, being consistent with tenacious binding of proteins.

– Albumin & lysozyme adsorbed to PS exhibit no unfolding enthalpy, whereas lysozyme to hydrophilic contact lens 50% enthalpy of native protein.

– Streptavidin adsorbed to PS displays unfolding enthalpy very similar to that for native protein in solu. because of greater stability.

* streptavidin-biotin complex uses in purification or detection of various biomolecules.

Biological Changes • Solution-phase fibrinogen does not bind the antibody

raised to fragment D, but surface-adsorbed fibrinogen does. RIBS(receptor-induced binding site) antibodies bind to fibrinogen only after the fibrinogen has bound to either a solid surface or to platelet IIb/IIIa receptor.

• Platelet adhesion to PMMA has been correlated with amount of antifibrinogen binding,

• suggesting that adsorbed fibrinogen is in different conformation on various PMMAs

• Fibrinogen undergoes a time-dependent transition that results in reduced platelet, antibody binding, and changes in amide II frequency

(1550cm-1). • Vitronectin undergoes conformational changes upon adsorption that affect its ability to bind heparin and its IR spec. (Ch.3.2: Fig.10)

Time-dependent transition

• On hydrophobic self-assembled monolayer, fibronectin has poor cell spreading for unless albumin is coadsorbed (“rescuing”); Albumin effect on fibronectin’s ability to promote cell spreading on PS.

• But Albumin should reduce the amount of adsorbed fibronectin, as it competes for sites on the surface. The adsorbed albumin keep the adsorbed fibronectin from undergoing structural changes.

• In part from differences in availability of epitopes on adhesive proteins for cell surface receptor.

• Both amount of adsorbed protein and its bioreactivity are actively influenced by properties of the surface.

Importance of Adsorbed Proteins • Table 1 summarizes the principle underlying the influence

of adsorbed proteins. 1. Synthetic foreign materials acquire bioreactivity only

after first interacting with dissolved proteins. (interaction of proteins, mediating cell adhesion)

2. Platelets are a major example 3. Sensitivity of platelets

a. Strongly adhesive proteins; fibrinogen, fibronectin,,, b. Concentrating, localizing, immobilizing effects of

proteins c. Receptors in platelets

4. Principles of protein adsorption a. Monolayer adsorption and competition b. Driving forces; intrinsic surface activity, proteins conc. c. Various selectivity of adsorption d. Biological activity on different surfaces

과제 : • Adsorbed protein의 구조와 기능 그리고 응용성을 정리

예) fibrinogen, fibronectin, vitronectin, blood proteins, steroid hormones, lipoproteins, growth factors, cytokines, etc .