2.79J/3.96J/BE.441/HST522J BIOMATERIALS-TISSUE INTERACTIONS: “Tools” for Understanding the Molecular, Cellular, and Physiological, Bases of the Tissue Response to Implants M. Spector, Ph.D. and I.V.M. Spector, Ph.D. and I.V. Yannas, Ph.D. Massachusetts Institute of Technology Harvard Medical School Brigham and Women’s Hospital VA Boston Healthcare System BIOMATERIALS-TISSUETISSUE INTERACTIONS Tissue* + Biomaterial** CellCell ++ Matrix** * Structure comprising cells of the same type ** Solid surface Page 1 In Tissue Cell ++ Extracellular MatrixMatrix In Tissue Engineering Scaffolds Cell + Biomaterial Scaffold CELL-MATRIX INTERACTIONS CONCEPTS FOR UNDERSTANDINGCONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS ? Control Volume ? Unit Cell Processes ? Types of Tissues ? Tissue Formation and RemodelingTissue Formation and Remodeling In Vitro ? Wound HealingWound Healing In Vivo Page 2 2 Cell Matrix Extracellular Articular Cartilage 5 3 1 4 6 40min Chondrocytes (P2 Canine) in a . Source: B. Kinner Type I Collagen-GAG Scaffold B. Kinner “Control Volume” Image removed due to copyright considerations Page 3 UNIT CELL PROCESSES Concept of a “Control Volume” around a Cell CellCell ++ MatrixMatrix ProductProduct ++ Soluble (Regulator)(Regulator) Regulator B “Control Volume” Soluble Regulator A UNIT CELL PROCESSES Concept of a “Control Volume” around a Cell CellCell ++ MatrixMatrix ProductProduct ++ Soluble (Regulator)(Regulator) Regulator B “Control Volume” Soluble Regulator A Mechanical Loading (Strain) Page 4 CONCEPTS FOR UNDERSTANDINGCONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS ? Control Volume ? Unit Cell Processes ? Types of Tissues ? Tissue Formation and RemodelingTissue Formation and Remodeling In Vitro ? Wound HealingWound Healing In Vivo UNIT CELL PROCESSES ? Mitosis ? Migration ? Synthesis ? Contraction ? Endocytosis ? Exocytosis Page 5 1mm 500mm COLLAGEN-GAG MATRICES: MODEL BIOMATERIALS (ANALOGS OF EXTRACELLULAR MATRIX) Investigation of cell interactions (UCPs ) in vitro ? Type I (bovine and porcine) ? Type II (porcine) ? Chondroitin 6-sulfate ? Freeze -dried ? Dehydrothermally cross-linked ? Additional cross-linking IV Yannas, et al. PNAS, 1989 Image removed due to copyright considerations. Image removed due to copyright considerations. CELLCELL –MATRIX INTERACTIONS WITHMATRIX INTERACTIONS WITH COLLAGEN-GAG MATRICESGAG MATRICES IN VITRO ? Can provide insights into interrelationshipsCan provide insights into interrelationships among cell processes. – How do mitosis and synthesis interrelate? – How do mitosis and synthesis relate toHow do mitosis and synthesis relate to contraction? – How does migration relate to contraction? ? Can provide insights into cell behaviorCan provide insights into cell behavior in vivo. ? Can provide insights into scaffold compositionCan provide insights into scaffold composition and structure for improved performance inand structure for improved performance in regenerative medicine. Page 6 Chondrocytes (Passage 2 Canine) in a Type I Collagen-GAG Matrix J. Cheng Live cell imaging for a period of 5 hours. Image removed due to copyright considerations. CELLCELL –MATRIX INTERACTIONS ? Mitosis ? Migration ? Synthesis ? Contraction Page 7 DNA Content Chondrocyte (P2 Canine) in a Type I Collagen-GAG Matrix: Mitosis J. Cheng 0 10 20 30 40 50 60 0 10 20 30 Day DNA (ug) DHT UV GTA EDAC CR Lee, et al., Biomat. 2001;22:3145 Mean – SEM Effects of Cross-Linking on Chondrocyte ProliferationLinking on Chondrocyte Proliferation in Collagen-GAG Matrices Page 8 CELLCELL –MATRIX INTERACTIONS ? Mitosis ? Migration ? Synthesis ? Contraction 40min Chondrocytes (P2 Canine) in a Type I Collagen GAG Matrix: Migration and Contraction B. Kinner Page 9 CELLCELL –MATRIX INTERACTIONS ? Mitosis ? Migration ? Synthesis ? Contraction Protein Synthesis; Proline Incorporation 0 0.05 0.1 0.15 2 7 15 29 Day nmolnmol /hour//hour/ mm g DNAg DNA Proteoglycan Synthesis; Sulfate Incorporation 0 0.01 0.02 0.03 2 7 15 29 Day DHT UV GTA EDC Effects of Cross-Linking on Chondrocyte BiosynthesisLinking on Chondrocyte Biosynthesis in Collagen-GAG Matrices CR Lee, et al., Biomat. 2001;22:3145 Page 11 CELLCELL –MATRIX INTERACTIONS ? Mitosis ? Migration ? Synthesis ? Contraction Chondrocytes (P2 Canine) in a Type I Collagen-GAG Matrix: Contraction Image removed due to copyright considerations. 40 min B Kinner Page 12 Cell-Mediated Contraction 21 days S. Vickers Image removed due to copyright considerations. Image removed due to copyright considerations. Image removed due to copyright considerations. Non-Seeded: 8 days Cell-Seeded: 8 days Non-Seeded and Cell-Seeded Collagen-GAG Scaffolds 30 40 50 60 70 80 90 100 0 10 20 30 Day % Original Diameter DHT UV GTA EDAC Mean – SEM CR Lee, et al., Biomat. 2001;22:3145 collagen low modulus high modulus Adult canine articular chondrocytes (passage 3) contract a type I -GAG matrix, reflected in the decrease in diameter Little x-link., Highly x-link., Page 14 Human Articular Chondrocytes in Monolayer Culture IH - Green: a -smooth muscle actin; Orange: type II collagen Image removed due to copyright considerations. Chondrocytes express the gene for a -smooth muscle actin and this enables them to contract B. Kinner, et al. JOR 2001;19:233 a-Smooth Muscle Actin Immunohistochemistry of Human Articular Cartilage Image removed due to copyright considerations. Kim and Spector, JOR 2000;18:749 Page 15 MUSCULOSKELETAL CELLS THAT CAN EXPRESSMUSCULOSKELETAL CELLS THAT CAN EXPRESS a-SMOOTH MUSCLE ACTIN AND CAN CONTRACT ? Articular chondrocyte ? Osteoblast ? Meniscus fibroblast andMeniscus fibroblast and fibrochondrocyte ? Intervertebral disc fibroblast anddisc fibroblast and fibrochondrocyte ? Ligament fibroblast ? Tendon fibroblast ? Synovial cell ? Mesenchymal stem cell M. Spector, Wound RepairWound Repair Regen. 9:11-18 (2001) POSSIBLE ROLES FORPOSSIBLE ROLES FOR a-SMOOTH MUSCLESMOOTH MUSCLE ACTIN-ENABLED CONTRACTION Musculoskeletal Connective Tissue Cells ? Tissue engineering Contracture of scaffolds ? Healing Closure of woundsClosure of wounds (skin wounds and bone fractures) ? Disease processes Contracture (Dupuytren’s) ? Tissue formation Modeling of ECM architecture and remodeling (e.g.,e.g., crimp in ligament/tendon?) Page 16 CONCEPTS FOR UNDERSTANDINGCONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS ? Control Volume ? Unit Cell Processes ? Types of Tissues ? Tissue Formation and RemodelingTissue Formation and Remodeling In Vitro ? Wound HealingWound Healing In Vivo TYPES OF TISSUES Which Tissues Can Regenerate Spontaneously? v ? Articular Cartilage,Articular Cartilage, Ligament, IntervertebralLigament, Intervertebral Disc, Others v Nerve v ? Smooth v? Cardiac, Skeletal Muscle v Epithelia (e.g., epidermis) v ? Bone Connective Tissues NoYes Page 17 CellCell + Matrix Connective Tissue Epithelia Muscle Nerve BIOMATERIALS-TISSUETISSUE INTERACTIONS UNIT CELL PROCESSES Concept of a “Control Volume” around a Cell CellCell ++ MatrixMatrix ProductProduct ++ Soluble (Regulator)(Regulator) Regulator B “Control Volume” Soluble Regulator A Mechanical Loading (Strain) Page 18 Cell ++ Matrix Connective Tissue Epithelia Muscle Nerve Adhesion Protein Collagen Biomaterial BIOMATERIALS-TISSUETISSUE INTERACTIONS BIOMATERIALS-TISSUETISSUE INTERACTIONS CellCell + Matrix Connective Tissue Epithelia Muscle Nerve Adhesion Protein Collagen Biomaterial Integrin Page 19 “UNIT CELL PROCESSES” Cell + Matrix Mitosis Synthesis Migration Contraction Endocytosis Exocytosis UCP Connective Tissue Epithelia Muscle Nerve “UNIT CELL PROCESSES” Cell + MatrixMatrix ProductProduct Mitosis Cell proliferation Synthesis Matrix molecules,Matrix molecules, enzymes, cytokines Migration Translocation Contraction Strain Endocytosis SolubilizedSolubilized fragments Exocytosis Regulators UCP Connective Tissue Epithelia Muscle Nerve Page 20 “UNIT CELL PROCESSES” Cell + MatrixMatrix Product ++ Regulator Regulator Mitosis Synthesis Migration Contraction Endocytosis Exocytosis UCP Connective Tissue Epithelia Muscle Nerve Cytokines (Growth Factors) “UNIT CELL PROCESSES” Cell + MatrixMatrix Product + Regulator Regulator Mitosis Synthesis Migration Contraction Endocytosis Exocytosis UCP Connective Tissue Epithelia Muscle Nerve Adhesion Protein Collagen Biomaterial Cytokines (Growth Factors) Integrin Mechanical Force (Strain) Page 21 “UNIT CELL PROCESSES” Cell + MatrixMatrix Product + Regulator Regulator Mitosis Synthesis Migration Contraction Endocytosis Exocytosis UCP Connective Tissue Epithelia Muscle Nerve Adhesion Protein Collagen Biomaterial Cytokines (Growth Factors) Integrin Mechanical Force (Strain) “UNIT CELL PROCESSES” Cell + MatrixMatrix Product + Regulator RegulatorRegulator (TGF-b 1) Mitosis Synthesis Migration Contraction Endocytosis Exocytosis UCP Connective Tissue Epithelia Muscle Nerve Adhesion Protein Collagen Biomaterial Cytokines (Growth Factors) Integrin Matrix strain (contracture/ shrinkage) Page 22 “UNIT CELL PROCESSES” Fibroblast + Collagen Contracture + Reg. TGF-b 1 Contraction CONCEPTS FOR UNDERSTANDINGCONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS ? Control Volume ? Unit Cell Processes ? Types of Tissues ? Tissue Formation and RemodelingTissue Formation and Remodeling In Vitro ? Wound HealingWound Healing In Vivo Page 23 TISSUE FORMATION -FGF-2 AND REMODELING Image removed due to IN VITRO copyright considerations. +FGF-2 Canine chondrocytes grown in a type II collagen-GAG Image removed due to scaffold for 2 weeks. copyright considerations. (Safranin O stain for GAGs ) N. Veilleux CONCEPTS FOR UNDERSTANDINGCONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS ? Control Volume ? Unit Cell Processes ? Types of Tissues ? Tissue Formation and RemodelingTissue Formation and Remodeling In Vitro ? Wound HealingWound Healing In Vivo Page 24 WOUND HEALING Roots of Tissue EngineeringRoots of Tissue Engineering Injury Inflammation (Vascularized tissue) Reparative Process Regeneration* Repair (Scar) CT: bone CT: cartilage Ep: epidermis: epidermis NerveNerve Muscle: smoothMuscle: smooth Muscle: cardiac,Muscle: cardiac, skel. 4 Tissue Categories Connective Tissue Epithelium Nerve Muscle *spontaneous RESPONSE TO IMPLANTS: WOUND HEALING Surgical Implantation Vascular Response Clotting Phagocytosis Neovascularization New Collagen Synthesis Tissue of Labile and Stable Cells Tissue of Permanent Cells Framework Framework Scarring IntactIntact Destroyed (fibrous encapsulation; synovium) Regen.. Scarring Chronic Inflammation (incorp.. (fibrous encapsulation; of implant) synovium) Chronic Inflammation AcuteAcute Inflammation Granulation Tissue Implant Movement Page 25