Massachusetts Institute of Technolog Technology Harvard Medical School Schoo Brigham and Womens Hospital VA Boston Healthcare System

2.79J/3.96J/20.441/HST522J

BIOMATERIALSBIOMATERIALS-TISSUE INTERACTIONS:
Tools for Understanding the M l l C ll l T l f U d di h Molecular, Cellular, and Physiological Bases of the Tissue Response to Implants p

M. Spector, Ph.D. Spector,

BIOMATERIALS-TISSUE INTERACTIONS

Tissue* + Biomaterial**

Cell

+ Matrix**

* Structure comprising cells of the same type ** Solid surface

CELL-MATRIX INTERACTIONS

In Tissu Tissue Cell + Extracellular Matrix In Tissue Engineering Scaffolds Cell + Biomaterial Scaffold

CONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS Control Volume Unit Cell Processes Types of Tissue Tissues Tissue Formation and Remodeling In Vitro Wound H li I Vi W d Healing In Vivo

Articular Cartilage

Extracellular Extracellular

Matrix

Cell

Figures by MIT OpenCourseWare.

4 mm

10 m

40min

Chondrocytes (P2 Canine) in a Type I Collagen-GAG Scaffold

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

2 3

Control Volume

4
B. Kinner

Zaleskas, J. M., et al. "Contractile forces generated by articular chondrocytes in collagen-glycosaminoglycan matrices." Biomaterials 25 no. 7-8 (2004):1299-1308.

UNIT CELL PROCESSES Concept of a Control Volume around a Cell

Soluble Regulator A

Cell + Matrix Matrix Cell Product + Soluble (Regulator) Regulator B (Regulator) Product
Control Volume

UNIT CELL PROCESSES Concept of a Control Volume around a Cell

Soluble Regulator

Autocrine factor

Cell + Matrix Cell + Matrix Product + Soluble (Regulator) (Regulator) Regulator Product
Control Volume Another Cell; Circulation; Paracrine Endocrine

UNIT CELL PROCESSES Concept of a Control Volume around a Cell

Soluble Mechanical Regulator Loading (Strain); Subject 2.785 A

Cell + Matrix Cell + Matrix Product + Soluble (Regulator) Regulator B (Regulator) Product
Control Volume

CONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS Control Volume Unit Cell Processes Types of Tissue Tissues Tissue Formation and Remodeling In Vitro Wound H li I Vi W d Healing In Vivo

UNIT CELL PROCESSES

Mitosis Migration Synthesis Contraction C i Endocytosis Exocytosis

UNIT CELL PROCESSES

Mitosis Migration Synthesis Contraction C i Endocytosis Exocytosis ? ?

UNIT CELL PROCESSES

Mitosis Migration Synthesis Contraction C i Endocytosis Exocytosis Apoptosis Differentiation

COLLAGEN-GAG MATRICES: MODEL BIOMATERIALS (ANALOGS OF EXTRACELLULAR MATRIX) Investigation of cell interactions (UCPs) in vitro Type I (bovine and porcine) T Type II ( (porcine) i ) Chondroitin 6-sulfate

1mm Freeze-dried Dehydrothermally cross linked cross-linked Additional cross-linking

See IV Yannas, et al. PNAS, 1989

500m

 CELL MATRIX INTERACTIONS WITH COLLAGEN-GAG MATRICES IN VITRO

Can provide insights into interrelationships among cell processes.
How do mitosis and synthesis interrelate? How do mitosis and synthesis relate to contraction? How does migration relate to contraction?

Can provide insights into cell behavior in vivo. Can provide insights into scaffold composition and structure for improved p p performance in regenerative medicine.

Chondrocytes (Passage 2 Canine) in a Type I Collagen-GAG Matrix Collagen GAG

Live cell imaging for a period of 5 hours.

J. Cheng

CELL MATRIX INTERACTIONS

Mitosis Migration Synthesis S th i Contraction

Chondrocyte (P2 Canine) in a Type I Collagen-GAG Matrix: Mitosis

Photo removed due to copyright restrictions.

J. Cheng

Effects of Cross-Linking on Chondrocyte Proliferation DNA Content in Collagen-GAG Matrices

60 50
Mean SEM

DN (ug NA g)

40 30 20 10 0 0 0 10 10 20 20

DHT UV GTA EDAC

Inc. crosscrosslinking

30 30

Day

CR Lee, et al., Biomat. 2001;22:3145

CELL MATRIX INTERACTIONS

Mitosis Migration Synthesis Contraction

Fibroblasts Migrate Away from Soft Substrates

NIH 3T3 cells are plated on polyacrylamide substrates with a transition in flexibility. The soft side is marked with fluorescent beads (to the left). Cells turn to avoid the soft substrate as they approach the boundary from the stiff side, by retracting the leading lamellipodium that crossed the boundary.

Courtesy of Yu-Li Wang. Used with permission.

C.-M. Lo, et al., Biophys. J. 79:144 (2000)

Fibroblasts Migrate Toward Stiff Substrates

NIH 3T3 cells are plated on polyacrylamide substrates with a transition in flexibility. The soft side is marked with fluorescent beads (to the left). Cells turn toward and enter the stiff side as they approach the boundary from the soft side, by expanding protrusions toward the boundary into a leading lamellipodium.

Courtesy of Yu-Li Wang. Used with permission.

C.-M. Lo, et al., Biophys. J. 79:144 (2000)

Fibroblasts Migrate Toward Stretching Forces

NIH 3T3 cells are plated on polyacrylamide substrates. Pulling forces are exerted by inserting a blunted needle in the substrate near the trailing end of the cell and dragging the needle away from the cell. Cells switch the direction of migration by expanding secondary protrusions toward the needle into a leading lamellipodium.

Courtesy of Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

C.-M. Lo, et al., Biophys. J. 79:144 (2000)

Fibroblasts Migrate Away from Compressing Forces

NIH 3T3 cells are plated on polyacrylamide substrates. Pushing forces are exerted by inserting a blunted needle in the substrate near the leading edge of an approaching cell and moving the needle toward the cell. Cells switch the direction of migration by retracting the leading lamellipodium.

Courtesy of Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

C.-M. Lo, et al., Biophys. J. 79:144 (2000)

Migration of Epithelial Cells In Vitro in a Wound Healing Assay

100 m
2001 L. C. Santy and J. E. Casanova. License CC BY-NC-SA. Published Published by The Rockefeller University Press Rockefeller University Press. http://dx.doi.org/10.1083/jcb.200104019

Monolayers were wounded by scraping.

LC Santy, J. Cell Biol. 154:599 (2001)

40min
J. Zaleskas, et al., Biomat. 25:1299 (2004)

Chondrocytes (P2 Canine) in a Type I CollagenGAG Matrix: Migration and Contraction

2 3

4 5
B. Kinner

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

Diagram removed due to copyright restrictions. Fig. i Mad i KidneyIInt. 41 (1992): 562. Fi 2 inM dri, Kid (1992) 562 Schematic of the modulation of microvascular endothelial cell phenotype during angiogenesis.

Madri, Kidney Int. 41:562 (1992)

CELL MATRIX INTERACTIONS

Mitosis Migration Synthesis Contraction

Effects of Cross-Linking on Chondrocyte Biosynthesis in Collagen-GAG Matrices 0.15 01

Protein Synthesis; p Proline Incorporation

DHT UV GTA EDC 0.03

Proteoglycan Synthesis; Sulfate Incorporation

nmol/h hour/ hour/g DNA

0.1

0.02

0.05

0.01

0 2 7 15 29

0 2 7 15 29

Day

Day
CR Lee, et al., Biomat. 2001;22:3145

CELL MATRIX INTERACTIONS

Mitosis Migration Synthesis Contraction

-smooth muscle actin-fusion peptide (SMA-FP) inhibits the tension exerted by lung fibroblasts on silicone substrates. After washing our of the FP, cells contract again.

See video at http://jcb.rupress.org/content/suppl/2002/05/03/jcb.200201049.DC1/1.html

Hinz B, et al., J Cell Biol 157:657 (2002)

Chondrocytes (P2 Canine) in a Type I Collagen-GAG Matrix: Contraction g

J. Zaleskas, et al., Biomat. 25:1299 (2004)

40 min
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

B Kinner

40 min 40 min

300 min
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

J. Zaleskas, et al., Biomat. 25:1299 (2004)

Non-Seeded: 8 days Non-Seeded: 8 days

Cell-Seeded: 8 days Cell-Seeded: 8 days

10mm 10 10mm
Non-Seeded and Cell-Seeded Collagen-GAG Scaffolds

21 days

S. Vickers

Courtesy of Scott Vickers. Used with permission.

Adult canine articular chondrocytes (passage 3)3) contract type I I Adult canine articular chondrocytes (passage contract a a type collagen-GAG matrix, reflected inin the decrease in diameter collagen-GAG matrix, reflectedContraction g Cell-Mediated the decrease in diameter , Cell Mediated

100

Mean SEM

% Origina Diam O al meter

90 80 70 60 50 40 30 0 10

Highly x-link., high modulus

DHT UV GTA EDAC

Little x-link., low modulus

Day

20

30

CR Lee, et al., Biomat. 2001;22:3145

Human Articular Chondrocytes in Monolayer Culture

IH - Green: -smooth muscle actin; Orange: type II collagen

Chondrocytes express the gene for -smooth muscle actin and this enables them to contract

Courtesy of John Wiley and Sons, Inc. Used with permission. Source: Kinner, B., and M. Spector. J Orthop Res 19 (2001): 233-241.

B. Kinner, et al. JOR 2001;19:233

-Smooth Muscle Actin Immunohistochemistry of Human Articular Cartilage

Neg. control
Courtesy of John Wiley and Sons, Inc. Used with permission. Fig 1a and b in Kim, A. C., and M. Spector. "Distribution of Chondrocytes Containing Alpha-smooth Muscle Actin in Human Articular Cartilage." J Orthop Res 18 (2000): 749.

Kim and Spector, JOR 2000;18:749

MUSCULOSKELETAL CELLS THAT CAN EXPRESS a a-SMOOTH MUSCLE ACTIN AND CAN CONTRACT

Articular chondrocyte Osteoblast Meniscus fibroblast and fibrochondrocyte Intervertebral disc fibroblast and fibrochondrocyte Ligament fibroblast Tendon fibroblast Synovial cell M. Spector, Mesenchymal stem cell esenchymal stem cell Mesenchymal Wound Repair Regen.
9:11-18 (2001) 9:11-

POSSIBLE ROLES FOR a-SMOOTH MUSCLE ACTIN-ENABLED CONTRACTION

Musculoskeletal Connective Tissue Cells Tissue engineering Contracture of scaffolds Healing Closure of wounds ( (skin wounds and bone fractures) ) Disease processes Contracture (Dupuytrens) Tissue formation Modeling of ECM architecture and remodeling (e.g., crimp in ligament/tendon?)

CONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS

Control Volume Unit Cell Processes Types of Tissue Tissues Tissue Formation and Remodeling In Vitro Wound H li I Vi W d Healing In Vivo

TYPES OF TISSUES Which Tissues Can Regenerate Spontaneously?

Yes Connective Tissues Bone Articular Cartilage, Ligament, Intervertebral Disc, Others Epithelia (e.g., epidermis) Muscle Cardiac, Skeletal Smooth Nerve No

BIOMATERIALS-TISSUE INTERACTIONS

Cell C ll + Matrix M t i
Connective Tissue Epithelia Muscle M l Nerve

UNIT CELL PROCESSES Concept of a Control Volume around a Cell

Soluble Mechanical Regulator Loading (Strain) A

Cell + Matrix Cell + Matrix (Regulator) (Regulator)

Product Product + Soluble Regulator B

Control Volume

BIOMATERIALS-TISSUE INTERACTIONS

Cell + Matrix
Connective Tissue Epithelia Muscle Nerve N

Adhesion Protein Collagen Biomaterial

BIOMATERIALS-TISSUE INTERACTIONS

Cell + Matrix
Connective Tissue Epithelia Muscle Nerve Adhesion Protein Collagen Biomaterial

Integrin

Chinese hamster ovary cell migration in a wound-healing assay

CHO cells express the 51 but not 41 integrin
C. Watters, Cell Biol. Ed. , 2:210 (2003) K.A. Pinco, Mol. Biol. Cell 13:3203 (2002)

Photos removed due to copyright restrictions. Figure 3 in Watters, C. Cell Biol. Ed. 2:210 (2003) Image and links to associated videos at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC256980/figure/F3/

Cells transfected with plasmid DNAs for 4 and 4/GFP

Migration of fibroblast-like fibrosarcoma cells in a 3-D collagen lattice

K. Wolf, et al., J. Cell Biol. 160:267 (2003)

Reduction of migration speed and induction of detached, nonmobile spherical morphology by adhesion perturbing anti1 i t i t bi ti 1 integrin antibody, as a consequence of impaired collagen fibril binding.

UNIT CELL PROCESSES

UCP
Mitosis Synthesis S nthesis Migration Contraction Endocytosis Exocytosis

Cell + Matrix
Connective Tissue Epithelia Muscle Nerve

UNIT CELL PROCESSES

UCP
Mitosis Synthesis y

Cell + Matrix
Connective Tissue Epithelia Muscle Nerve N

Product

Cell proliferation Matrix molecules, , enzymes, cytokines Migration Translocation Contraction Strain Endocytosis Solubilized fragments Exocytosis Regulators

UNIT CELL PROCESSES C OC SS S

Regulator UCP

Cell + Matrix
Connective Tissue Epithelia Muscle N Nerve

Product + Regulator
Mitosis Synthesis Migration Contraction Endocytosis Exocytosis Cytokines (Growth Factors)

REGULATORS
Cytokines/Growth Factors
http://themedicalbiochemistrypage.org/growthhttp://themedicalbiochemistrypage.org/growthfactors.html (previously: http://web.indstate.edu/thcme/mwking/growthhttp://web.indstate.edu/thcme/mwking/growthfactors.html

http://www.copewithcytokines.de/

UNIT CELL PROCESSES C OC SS S

Regulator Mechanical Force (Strain) UCP

Cell Ce + Matrix at

Product + R egu ato oduct egulator

Connective Adhesion Mitosis Tissue ssue Protein Synthesis Epithelia Collagen Migration Muscle Biomaterial Contraction Endocytosis Nerve Integrin Exocytosis

Cytokines (Growth Factors)

UNIT CELL PROCESSES C OC SS S

Regulator Mechanical Force (Strain) UCP

Cell Ce + Matrix at

Product + R egu ato oduct egulator

Connective Adhesion Mitosis Tissue ssue Protein Synthesis Epithelia Collagen Migration Muscle Biomaterial Contraction Endocytosis Nerve Integrin Exocytosis

Cytokines (Growth Factors)

UNIT CELL PROCESSES C OC SS S

Regulator (TGF-1) (TGFUCP

Cell + Matrix

Product + Regulator

Matrix strain Cytokines Mitosis Connective Adhesion Synthesis (contracture/ (Growth Factors) Tissue Protein g ) Migration shrinkage) Epithelia E ith li Collagen Muscle Biomaterial Contraction Endocytosis Nerve Exocytosis Integrin

UNIT CELL PROCESSES C OC SS S

TGFTGF-1 G

Contraction

Fibroblast + Collagen

Contracture + Reg.

CONCEPTS FOR UNDERSTANDING BIOMATERIALS - TISSUE INTERACTIONS Control Volume Unit Cell Processes Types of Tissue Tissues Tissue Formation and Remodeling In Vitro Wound H li I Vi W d Healing In Vivo

+FGF-2

TISSUE FORMATION AND REMODELING IN VITRO

+FGF-2
Canine chondrocytes grown in a type II collagen-GAG scaffold for 2 weeks. (Safranin O stain for GAGs)

N. Veilleux, et al., Osteoart. & Cart. 2005;13:278

Courtesy of Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

CONCEPTS FOR UNDERSTANDING BIOMATERIALS-TISSUE INTERACTIONS Control Volume Unit Cell Processes Types of Tissue Tissues Tissue Formation and Remodeling In Vitro Wound H li I Vi W d Healing In Vivo

WOUND HEALING Roots of Tissue Engineering

Injury Inflammation (Vascularized tissue) Reparative R ti Process Regeneration* CT: bone Ep: epidermis epidermis Muscle: smooth
*spontaneous
4 Tissue Categories Connective Tissue Epithelium Nerve Muscle

Repair (Scar) CT: cartilage Nerve Muscle: cardiac, skel.

RESPONSE TO IMPLANTS: WOUND HEALING

Surgical Implantation Acute Inflammation Vascular Response Clotting Phagocytosis Neovascularization New Collagen Synthesis
Implant Movement

Granulation Tissue

Tissue of Labile and Stable Cells Framework F F k Framework k Intact Destroyed

Tissue of Permanent Cells Scarring S i (fibrous encapsulation; synovium) Chronic Inflammation

Regen. Scarring (incorp. (fibrous encapsulation; of implant) synovium) Chronic Inflammation

MIT OpenCourseWare http://ocw.mit.edu

20.441J / 2.79J / 3.96J / HST.522J Biomaterials-Tissue Interactions

Fall 2009

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