an introduction to tissue engineeringreference: cover page of science, volume 276, issue 5309 (4...

MATERIAL-RELATED APPROACHES TO PROMOTE

CELL FUNCTIONS PERTINENT TO NEOTISSUE FORMATION

FOR BIOMEDICAL APPLICATIONS

Rena Bizios, Ph.D.

Peter T. Flawn Professor

Department of Biomedical Engineering

The University of Texas at San Antonio

San Antonio, TX 78249

USA

A Perspective of

Past Accomplishments and Current Advances

regarding

Biomaterials for Prosthetic Devices

as well as

some Thoughts and Comments regarding

Future Trends

pertinent to

Implant Biomaterials

MEDICAL NEEDS

Need for substitutes for limbs, tissues and organs result from:

birth defects

trauma

age-related diseases

degenerative conditions

end-stage organ failure

etc.

TISSUE/ORGAN GENERATION IN BIOLOGY

* Reference: http://www.nidr.nih.gov/spectrum/NIDCR4/4grasec2.htm

EXAMPLES OF TISSUE REGENERATION *

Limbs and tails of salamanders and newts Antlers of deer

• Regeneration of tadpole tails

• Plugging holes in bat wing membranes

• Filling-in ear holes in rabbits, domestic cats, and bats

• Regrowth of the tips of mouse toes

* Reference: Cover page of Science, Volume 276, Issue 5309 (4 April 1997)

The Myth of Prometheus*

• Prometheus stole fire from the

Gods of Olympus and gave it to

humans.

• Prometheus was punished by the

gods to have a portion of his liver

eaten each day by an eagle.

• The liver regenerated overnight

providing the eagle with an endless

food supply, and Prometheus with

eternal torture.

ORGAN REGENERATION IN HUMANS

TISSUE REGENERATION IN HUMANS IS EXTREMELY LIMITED

Special cases:

Liver

Bone

MATERIALS USED FOR PROSTHETIC DEVICES

PAST ….TO PRESENT

DEVELOPMENT OF BIOMATERIALS

• In the past, “ordinary” materials were used for prosthetic devices

http://humanrestore.com/wp-content/uploads/2011/02/wooden_toes

_from_egyptian_mummy_37143800.jpg

Reference: As found in “dvd.monstersand critics.com”

EXAMPLES OF ANCIENT AND LEGENDARY PROSTHETIC DEVICES

Reference: Cover page of Science, Volume 295, Issue Number 5557, pg. 995 (8 February 2002)

DEVELOPMENT OF PROSTHETIC DEVICES

http://www.humanpl.us/wp-

content/uploads/2009/12/dean-kamen-arm.jpg 2009

Reference: Cover page of Science, Volume 295,

Issue Number 5557, pg. 995 (8 February 2002)

Reference: Science, Volume 295, Issue Number 5557, pg. 995 (8 February 2002)

DEVELOPMENT OF PROSTHETIC DEVICES (continued)

Reference: Advertisement in press (2007)

RECENT DEVELOPMENTS

OSCAR PISTORIUS MAKES OLYMPIC HISTORY (400 meter qualifying heat; August 4, 2012)

2012 OLYMPIC GAMES, LONDON, UNITED KINGDOM

Building

http://www.bbc.co.uk/news/

(August 4, 2012)

Types of Prostheses

• Mostly External

• Mechanical

• Electrical

• Temporary

• Intermittent Use

“TRADITIONAL” PROSTHETIC DEVICES

Reference: Cover page of Science, Volume 295,

Issue Number 5557 (8 February 2002)

Characteristics/Specifications

• Mechanical

• Electrical

• “Combination”

• Internal

• Long-term

IMPLANTED PROSTHESES/DEVICES

MATERIALS IN IMPLANTED PROSTHESES/ DEVICES

PAST…..TO PRESENT

DEVELOPMENT OF BIOMATERIALS

• In the past, “ordinary” materials were used for prosthetic devices

• In the past, synthetic materials were chosen and used for various

biomedical applications by trial-and error approaches

http://www.marketmanila.com/archives/s

ausage-casings-a-k-a-pigs-intestines

http://www.buycheapr.com/us/result.jsp?q=women+girdles

EXAMPLES OF “ORDINARY” MATERIALS USED FOR EARLY BIOMEDICAL APPLICATIONS

The first family of hollow-fiber dialyzers

(C-DAK, “Cordis Dow Artificial Kidney“)

http://www.fmc-ag.com/262.htm 2011

http://www.shubhanmedical.com/wp-content/

uploads/2010/09/MAQUET_Graft3.jpg

EXAMPLES OF CURRENT COMMERCIALLY AVAILABLE DIALYZERS AND VASCULAR GRAFTS

DEVELOPMENT OF BIOMATERIALS

• In the past, synthetic materials were chosen and used for

biomedical applications by a trial-and error approach.

• Eventually, such materials were designed to be

non-toxic

non-pyrogenic

non-allergenic, etc.

BUT STILL

they did not produce desired and controlled interactions

with the surrounding tissues, and

they did not trigger the normal healing process of tissues at the implant-tissue interface.

Contrôle Corail seul

16 weeks 16 weeks 16 weeks 16 weeks4 weeksPost-operative

Résultats RadiographiquesCLINICAL EXAMPLE: Nonunion Bone Defects

Slide courtesy of Dr. A. Meunier

16 weeks 16 weeks Post-Operative 4 weeks

EXAMPLES OF BONE IMPLANTS

Slide courtesy of

Dr. A. Meunier

orthopaedic (hip prosthesis dental

http://www.siteadmincp.com/

sites/site135/

site_images/IMPLANT_TOOTH.jpg

BIOMEDICAL NEED AND CHALLENGE

How to promote tissue regeneration and formation

of normal, functional new tissue in vitro and in vivo.

RATIONALE

for

RECENT APPROACHES and DEVELOPMENTS

THE INJURED TISSUE SITE: WOUND HEALING PROCESS

T I S S U E I N J U R Y

Biological Fluids (blood; interstitial fluids)

Proteins

Cells

Tissues

Biological Fluids (blood; interstitial fluids)

Proteins

Cells

Tissues

THE TISSUE/IMPLANT INTERFACE

Cells

Extracellular Matrix

Adhesive Proteins

Growth Factors

Cytokines

Biological Fluids

Devices

Synthetic Materials

Scaffolds

I

N

T

E

R

F

A

C

E

COMPARISON

Biological Tissues

Cells

Extracellular Matrix

Adhesive Proteins

Growth Factors

Cytokines

Cues/Stimuli

(biochemical/biophysical)

Cells

Biomaterials

Ceramics

Metals

Plastics

Cues/Stimuli

(biochemical/biophysical)

“Engineered” Tissues

I

N

T

E

R

F

A

C

E

ILLUSTRATION: PREPARATION OF A TISSUE ENGINEERING CONSTRUCT

Cell seeded scaffolds

Slide courtesy of Dr. C.M. Agrawal, UTSA

RECENT DEVELOPMENTS

Bioinspired and Biomimetic Aproaches

Recent research activities have focused on engineering

synthetic biomaterials for bio-recognition (by cells and proteins)

and for specificity in order to achieve precise, defined , and timely

biological responses.

TISSUE MICROENVIRONMENT* BY DESIGN

*This term was adapted from: Lumelsky, N. L., “Commentary: Engineering of Tissue Healing and Regeneration”.

Tissue Engineering 13: 1393-1398 (2007).

THE TISSUE/IMPLANT INTERFACE

Devices

Synthetic Materials

Scaffolds

Biological Fluids (blood; interstitial fluids)

Proteins

Cells

Tissues

I N T E R F A C E

MECHANISMS OF MAMMALIAN CELL ADHESION

ON

SUBSTRATE SURFACES

CELL FUNCTIONS PERTINENT TO

THE WOUND HEALING/TISSUE REGENERATION PROCESSES

• Adhesion (to substrates and to other cells)

• Morphology Changes

• Survival and Apoptosis

• Motility/haptotaxis

• Proliferation

• Synthesis and Deposition of an Extracellular Matrix

• Differentiation (if applicable)

• Cell-to-cell signaling

• etc.

PROTEIN-BIOMATERIAL INTERACTIONS

Biomaterial

Proteins

Schematics courtesy of Professor D.A. Puleo,

University of Kentucky, Lexington, KY

Cells

Biomaterial

CELL ADHESION ON BIOMATERIALS

BIOMATERIAL-RELATED APPLICATIONS

PROMOTION OF DIRECTED MAMMALIAN CELL ADHESION

ON PATTERNED SURFACES

MODIFIED CHEMICALLY WITH PROTEINS

EXPERIMENTAL APPROACHES: MICROCONTACT PRINTING

N1[3-(trimethoxysilyl)propyl]diethylenetriamine (DETA)

Octadecyltrichlorosilane (OTS)

Lattice Parameter, dLP 25, 35, 50 mm

Feature Width, w 7.9, 21.7, 43.3 mm

Coverage Fraction, h 10, 25, 50, 75%

dLP

w

Reference: Kam, L., W. Shain, J.N. Turner, R. Bizios, Biomaterials 20: 2343-2350 (1999).

CONTROLED ASTROGLIAL ADHESION ON MICROPATTERNED SURFACES

Reference: Kam, L., Doctoral Thesis, Rensselaer Polytechnic Institute, Troy, NY (1999).

PROMOTION OF DIRECTED MAMMALIAN CELL ADHESION

ON PATTERNED SURFACES

MODIFIED CHEMCIALLY WITH PEPTIDES

Extracellular Domain

Cytoplasm

Adapted from: Akiyama, S.K., K.M. Yamada, J. Biological Chemistry 260: 4492-4500 (1985)

HS-PG = Heparin Sulfate Proteoglycans

Plasma Membrane

SCHEMATIC ILLUSTRATION OF THE OSTEOBLAST ADHESION MECHANISMS

Octadecyltrichlorosilane (OTS)

N1[3-(trimethoxysilyl)propyl]diethylenetriamine (DETA);

further modified with either RGDS or KRSR.

L = 10 mm

d = 10, 50, 100, or 200 mm L

L

L

L

d

MICROPATTERNED SURFACES CHEMICALLY MODIFIED WITH IMMOBILIZED PEPTIDES

OSTEOBLAST CLUSTERING DEPENDENTS ON THE DIMENSIONS OF THE

MICROPATTERNS MODIFIED WITH KRSR

d = 10 mm d = 50 mm

d = 100 mm d = 200 mm

Reference: Hasenbein, M.E., T.T. Andersen, R. Bizios, Biomaterials 23: 3937-3942 (2002).

100 mm

CELL ADHESION ON SUBSTRATES MODIFIED WITH IMMOBILIZED PEPTIDES

Light Micrographs

Reference: Hasenbein, M.E., T.T. Andersen, R. Bizios, Biomaterials 23: 3937-3942 (2002).

Fluorescence

Micrographs Light Micrographs

Fibroblasts

Osteoblasts

Arginine-Glycine-Aspartic acid-Serine (RGDS) Lysine-Arginine-Serine-Arginine (KRSR)

Fluorescence

Micrographs

NEW SCIENTIFIC DIRECTIONS

MICROENVIRONMENT BY DESIGN

Devices

Synthetic Materials

Scaffolds

Biological Fluids (blood; interstitial fluids)

Proteins

Cells

Tissues

I N T E R F A C E

DEFINITIONS

• Nanophase/Nanostructured are materials characterized by

atomic domains spatially confined (grain size)

to less than 100 nm in at least one direction.

• Conventional (micron-size) are materials with grain sizes

greater than 100 nm.

Reference for the schematic:

Silver, F.H., D.L. Christiansen,Biomaterials Science and Biocompatibility,

Springer: New York, p.110, 1999.

Grain Growth

Crystal Nucleation

Grain boundary

impingement

EXCITEMENT

AND

POTENTIAL

NANOSTRUCTURED BIOMATERIALS PROMOTE SPECIFIC

FUNCTIONS OF SELECT MAMMALIAN CELLS

ENHANCED OSTEOBLAST ADHESION ON NANOPHASE CERAMICS

0

1000

2000

3000

4000

1 2 3 4

Grain Size (nm)

Cell

Den

sit

y

(cell

s/s

qu

are

cm

)

Alumina

24 4520 97

Titania

39 179

Hydroxylapatite

132 67

* * *

167 45

Glass

*

Culture media : DMEM supplemented with 10% fetal bovine serum.

Values are mean ± SEM; n = 3;

*p<0.01 (student t-test compared to respective conventional grain size ceramic).

Reference: Webster, T.J., C. Ergun, R.H. Doremus, R.W. Siegel, R. Bizios. Biomaterials 21: 1803-1810 (2000)

COMPARISON OF CELL ADHESION ON NANOPHASE ALUMINA

0

1000

2000

3000

4000

Osteoblasts Fibroblasts Endothelial Cells

Ce

ll D

en

sit

y

(c

ells

/sq

ua

re c

m)

Glass (reference substrate)

167 nm grain size alumina

45 nm grain size alumina

24 nm grain size alumina

*

*

**

*‡

‡

Glass (reference substrate)

Culture medium = DMEM supplemented with 10% fetal bovine serum.

Values are mean ± SEM; n = 3; *p<0.01 (student t-tests compared to 167 nm grain size alumina);

‡ p<0.01 (student t-tests compared to fibroblast and endothelial cell adhesion

on respective grain size alumina).

Reference: Webster, T.J., C. Ergun, R.H. Doremus, R.W. Siegel, R. Bizios. JBMR 51: 475 - 483 (2000)

0

1000

2000

3000

4000

Osteoblasts Fibroblasts Endothelial Cells

Ce

ll D

en

sit

y

(c

ells

/sq

ua

re c

m)

Glass (reference substrate)

167 nm grain size alumina

45 nm grain size alumina

24 nm grain size alumina

*

*

**

*‡

‡

Glass (reference substrate)

COMPARISON OF CELL ADHESION ON NANOPHASE ALUMINA

Culture medium = DMEM supplemented with 10% fetal bovine serum.

Values are mean ± SEM; n = 3;

*p<0.01 (student t-tests compared to 167 nm grain size alumina);

‡ p<0.01 (student t-tests compared to fibroblast and endothelial cell adhesion on respective

grain size alumina).

Reference: Webster, T.J., C. Ergun, R.H. Doremus, R.W. Siegel, R. Bizios. JBMR 51: 475 - 483 (2000).

QUESTION

How are cell functions modulated by nanoscale-size features

on material surfaces?

PROTEIN-BIOMATERIAL INTERACTIONS

Biomaterial

Proteins

Schematics courtesy of Professor D.A. Puleo, University of Kentucky, Lexington, KY

Borosilicate glass 167 nm alumina 24 nm alumina

Reference: Webster, T.J., C. Ergun, R.H. Doremus, R.W. Siegel, R. Bizios. JBMR 51: 475-483 (2000).

Values are ± SEM; n = 3; *p<0.01 (compared to 167 nm alumina pretreated with the respective

protein);

‡ p<0.01 (compared to respective grain size alumina pretreated with albumin)

PROTEIN ADSORPTION ON ALUMINA

Cells

Biomaterial

CELL ADHESION ON BIOMATERIALS

Schematic courtesy of Professor D.A. Puleo, University of Kentucky, Lexington, KY

OSTEOBLAST ADHESION ON SELECT PROTEINS PRE-ADSORBED ON

NANOPHASE ALUMINA

Cell culture medium = DMEM without serum. FBS= fetal bovine serum.

Substrate were pretreated with protein (0.5 mg/ml).

Values are mean ± SEM; n = 3; *p< 0.01 (student t-tests compared to 167 nm grain size alumina);

‡ p< 0.01 (student t-tests compared to respective grain size alumina pretreated with albumin).

Reference: Webster, T.J., C. Ergun, R.H. Doremus, R.W. Siegel, R. Bizios. JBMR 51: 475-483 (2000)

0

1000

2000

3000

4000

1 2 3 4 5 6

Protein

Series1

Series2

167 nm grain size alumina

24 nm grain size alumina

Cell D

en

sit

y

(cells/s

qu

are

cm

)

*‡

*‡

*‡ *

‡

Albumin10% FBS Laminin Denatured

Collagen

Fibronectin Vitronectin

Glass (reference substrate)

CONCLUSIONS

• Nanoscale features and/or grain sizes modulate the

type,

amount, and

conformation

of select adsorbed proteins.

• These protein interactions subsequently promote cell functions

(such as adhesion, etc.) of specific cells (selectivity).

CONCERNS

regarding

NANOSTRUCTURED MATERIALS:

BIOSAFETY

Need to determine any detrimental effects of nanostructured materials

at the following levels:

• gene

• subcellular

• cellular

• tissue

• organ

• whole body (systemically)

EVOLVING IDEAS FOR

NOVEL MATERIALS/BIOMATERIALS:

SCIENTIFIC DIRECTIONS OF PROMISE

http://www.pbs.org/wgbh/nova/tech/making-stuff-smarter.html

MATERIALS RESPONSIVE TO ENVIRONMENTAL CONDITIONS (such as temperature, pH, etc.)

SHAPE-MEMORY ALLOYS: Thermosensitive Materials

A material made from the pig small intestinal submucosa (SIS)

EXAMPLE OF A BIO-DERIVED EXTRACELLULAR MATRIX (ECM)

http://www.nibib.nih.gov/publicPage.cfm?section=gallery&action=desc&page=3&photo=6

Image courtesy of Dr. Stephen Badylak, University of Pittsburgh. Grant EB000506

Optical microscope images of a polymer nanocomposite taken (a) 2, (b) 10, (c) 20,

(d) 40, (e) 60 and (f) 90 seconds after a cut is made on the surface of the nanocomposite.

http://www.deakin.edu.au/itri/cmfi/research/areas/self-healing.php

SELF-HEALING POLYMERS AND NANOCOMPOSITES: AN EXAMPLE

PROTEIN-ENGINEERED BIOMATERIALS*

*Reference: Sengupta, D. S.A. Heilshorn. “Protein-Engineered Biomaterials: Highly tunable tissue engineering

scaffolds”. Tissue Engineering, Part B 16, 285 (2010).

FIG 2. Schematic of the iterative protein-engineered biomaterial design strategy.

(1) Design a DNA template for the protein engineered biomaterial

(2) Encode into a recombinant plasmid

(3) Use the plasmid to transfect the host cell of choice,

(4) Translate the genetic message and express the engineered protein

(5) Purify the protein

(6) Process to fabricate a protein engineered scaffold

(7) The scaffold is analyzed using in vitro and in vivo methods,

providing information about how to improve the properties

of the scaffold, thus prompting biomaterial redesign and

modification of the encoding DNA template.

1

2 3

4

5 6

7

NEW SCIENTIFIC DIRECTIONS

Reference: Cover page of Science, Volume 276,

Issue Number 5795 (29 September 2006)

BIOMATERIALS AS

DELIVERY VEHICLES FOR:

• STEM CELLS

• GROWTH FACTORS

• GENES

References: Gerstenfeld, L.C., Cullinane, D.M., Barnes, G.L., Graces, D.T., Einhorn, T.A., J Cell Bioc 88:873-874 (2003)

STAGES OF BONE FRACTURE REPAIR

LIMITATIONS, CHALLENGES, AND OPPORTUNITIES

SCHEMATIC ILLUSTRATION OF THE COMPONENTS AND STRUCTURE OF BONE

Reference: http://www.octc.kctcs.edu

CURRENT LIMITATIONS IN TISSUE ENGINEERING

• Vascularization

• Innervation

SCIENTIFIC CHALLENGES AND RESEARCH OPPORTUNITIES

• The current knowledge of the biology/physiology of functional tissue

formation is incomplete but is critically needed.

• Improved and/or thorough understanding of the wound healing

process in health and in disease is needed.

• The function of cells (including stem cells) pertinent to new tissue

formation needs to be elucidated and incorporated in implant

biomaterials.

.

CLOSING REMARKS

• The field of implant biomaterials is at another “historic cross-road”of

potential and promise.

• Further developments require establishment of scientific

fundamentals pertinent both to implant biomaterials and to

cells/tissues/organs.

• Success requires interdisciplinary, multidisciplinary, creative, and

novel approaches which incorporate cutting-edge knowledge of

processes and conditions pertinent to physiology and pathology.

ACKNOWLEDGMENTS

Former Graduate Students

at Rensselaer Polytechnic Institute

Dr. Kay C Dee

Ms. Meredith E. Hasenbein

Dr. Lance C. Kam

Dr. David A. Puleo

Dr. Thomas J. Webster

Colleagues

Ms. C. Charniga and Dr. H. Kimelberg, Department of Neurosurgery, Albany Medical College, Albany, NY.

Dr. J.N. Turner and Dr. W. Shain, Wadsworth Labs, NY State Health Department, Albany, NY.

Dr. T.T. Andersen, Department of Biochemistry, Albany Medical College, Albany, NY.

Dr. R.W. Siegel and Dr. R.H. Doremus, Department of Materials Science & Engineering, Rensselaer Polytechnic Institute, Troy, NY.

Nanophase Technologies, Inc., provided the nanophase alumina and titania

powders used in the respective studies of nanostrucutred ceramics

THANK YOU

for

YOUR ATTENTION

CASE STUDY*

• L.S., a 66-year old man, had a piece (about three-eights of

an inch long) cut off his right, middle finger by a gas-powered

model airplane propeller.

• The injury was treated using ACell Powder Wound Dressing

(ACell, Inc., Jessup, MD) every two-days. This powder (a

substance derived from pig bladder extract) had been used

for healing and tissue regeneration in animals (specifically,

horse ligaments) and had also received federal government

approval for use in humans.

• The finger regained its original length within 4 weeks.

• The finger looked like the normal finger within 4 months.

• A year later, the finger felt calloused but had tactile

sensation; it had a slight scar at the end; the nail grew at

twice the speed of the other finger nails.

* Reference:www.mysanantonio.com/news/metro/stories/MYSA0