Engineering Biopolymeric

Nanostructured Fibers And Films

For Tissue Engineering Applications

Beatriz Quiñones-Colón1

David Castilla2, Jorge

Almodóvar2

University of Puerto Rico

Mayaguez Campus 1Department of Biology

2Department of Chemical

Engineering

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For example, in the case of osseous tissue, common sources of lesions:

The partial or total loss of organ tissue or function has become one of the most

aggravating problems of human health.

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS

Osteonecrosis

Tumors Traumas

Problem:

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Bioinspiration: The Cell’s Microenvironment

MECHANICAL (Stiffness)

Cellular

Processes

Adhesion

Proliferation

Migration

Differentiation

BIOCHEMICAL (Cytokines ie. growth factors)

Extra-cellular

Matrix (ECM)

(Polysaccharides,

Proteins)

Tissue formation

Tissue regeneration

ANISOTROPY (Cues in gradients)

Need for Biomaterials to capture the native ECM

1. Provide Mechanical & Biochemical Cues

2. Fibrous Nature

Artwork from A. Kawska

Courtesy of C Albiges-Rizo

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 3

Our Approach: Polymeric Biomaterials

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Layer-by-Layer

(LbL) Films

Electrospun

Nanofibers

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 4

1) 2)

ELECTROSPINNING

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 5

Electrospun Scaffolds: Towards ECM

Mimetic Systems

Composition

Scale

Diameter

Morphology

Geometry

Orientation

To produce polymeric fibers of single or mixed materials. Uses a difference in potential to draw very fine (typically on the nano or micro scale)

fibers from a liquid (immediate desolvation).

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 6

Versatile technique used to mimic the extracellular matrix of tissues in

its:

Fiber diameter:

• Range = 49 – 140 nm

• Average = 77 nm

Electrospun Collagen Nanofibers

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 301(9), 2016, 1064-1075

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 7

Controlling Fiber Diameter

Voltage (25 – 45 kV) Flow Rate (0.5 – 3.0 mL/h)

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 8

Collagen fiber diameter can be tuned by:

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 301(9), 2016, 1064-1075

Oriented / Aligned Collagen Fibers

The body contains three

types of muscle tissue: (a)

skeletal muscle, (b)

smooth muscle, and (c)

cardiac muscle. (Same

magnification) Oriented/aligned fibers that mimics the native muscle

tissue. Spun at 5 mL/hr, 47 kV

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 9

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 301(9), 2016, 1064-1075

Glutaraldehyde Crosslinking Preserves

Nanofiber Structure

Crosslinked nanofibers Crosslinked nanofibers after

exposure to water at 37 °C

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 10

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 301(9), 2016, 1064-1075

Tunable Mechanical Properties by

Glutaraldehyde Crosslinking

Immersion

Vapor

Young's Modulus (MPa) UTS (MPa) Elongation at Break (%)

Immersion 4.1 ± 0.5 1.0 ± 0.4 29 ± 15

Vapor 2.7 ± 0.7 0.9 ± 0.2 38 ± 7

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 11

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 301(9), 2016, 1064-1075

Process preserves Collagen’s native

structure

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 12

FTIR reveals a preservation of collagen’s structure after

electrospinning.

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 301(9), 2016, 1064-1075

Collagen Nanofibrous Scaffold Supports

Mammalian Cell Growth

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 13

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 301(9), 2016, 1064-1075

Collagen Nanofibrous Scaffold Supports

Mammalian Cell Growth

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 14

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 301(9), 2016, 1064-1075

15 INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS

In this work, we successfully produced type I collagen nanofibers by

electrospinning, using a non-toxic solvent.

We observed that the solution preparation, electrospinning process, and

crosslinking methods do not affect the secondary structure of

the type I collagen used. Crosslinked membranes were stable in an

aqueous environment at body temperature, which is crucial for implants and

tissue regeneration purposes.

We demonstrated that it is possible to control the diameter of the

nanofibers by varying the voltage and the flow rate.

According to our results, membranes obtained have mechanical

properties in the range of native tissues and support

mammalian cell culture, indicating potential promise in field of the

tissue engineering.

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INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS

LAYER-BY-LAYER

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Polysaccharide-Based Layer-by-Layer Films

Chitosan

Heparin

LbL nano-film preparation on silicon substrate

Simplified molecular representation of LbL film

Takes advantage of the

electrostatic forces of

polymers to generate

multilayers over

biomaterial surfaces.

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS 17

LBL film chemistry and growth

• IR-VASE allows the

evaluation of film

chemistry and

growth.

Incre

asin

g n

um

be

r o

f la

ye

rs

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0

20

40

60

80

100

120

140

160

180

3 Bilayers 6 Bilayers 9 Bilayers 12 Bilayers Flu

ore

scen

ce M

easu

rem

ent

Number of Bilayers

Viability of NIH3T3 cell line cultured over chitosan-heparin bilayers

Day 1

Day 3

Proliferation of cells after one and three days of being

seeded onto bilayers.

Cells on six bilayers of CHI-HEP

SEM Images. Cells on Six bilayers

of CHI-HEP.

Proliferation of NIH3T3 Cell Lines with CHI-HEP Bilayers

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS

In conclusion, we can create polysaccharides based thin films by

the layer-by-layer method and we can control cell behavior in

these films.

In this work it was demonstrated that by applying the layer-by-

layer method and electrospinning, it is possible to replicate the

native extracellular matrix of tissues,

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Substrato

Superficies bioactivas

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Acknowledgments

Collaborators

UPRM

Dr. Aldo Acevedo

Dr. Barbara Calcagno

Dr. Madeline Torres

Dr. Paul Sundaram

Integra

Dr. Anibal Quintana

www.uprm.edu/biomaterials

http://www.uprm.edu/biomaterials

http://www.uprm.edu/p/biomaterials/graduate_students

Location Component Pure

collagen %

Nanofibers % Crosslinked

nanofibers %

1613.2 β-turns 16.49 18.61 18.78

1626.4 β-sheets 21.09 26.27 19.68

1637.3 triple helix 18.92 21.01 18.54

1650.6 unordered 31.11 23.28 29.84

1655.0 α-helix 12.40 10.85 13.17

Preservation of Collagen’s Secondary Structure

Pure Nanofibers Crosslinked

Castilla D., Almodovar J. et al, Macromol. Mater. Eng., 2016, 10.1002/mame.201600156

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS

Infrared Variable Angle Spectroscopic

Ellipsometer (IR-VASE)

https://www.google.com.pr/search?q=ir+vase+polarized+light&espv=2&biw=112

3&bih=701&source=lnms&tbm=isch&sa=X&ved=0ahUKEwimyqX7po3MAhVCq

B4KHccNCpMQ_AUIBigB#imgrc=2WdQwbMg_RDGlM%3A

Chemical Composition

Sample thickness

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS

This a non-destructive characterization technique combining the fundamentals of

ellipsometry and FTIR spectroscopy.

Electrospinning of type I Collagen

1. Solvent

90% acetic acid in water

2. Concentration

20% w/v

3. Voltage

47 kV

4. Flow Rate

1 mL/hr

5. Gluteraldehyde

crosslinking

Immersion

Vapor

INTRODUCTION OBJECTIVES METHODOLOGY RESULTS CONCLUSIONS