silkfibroin - unitrento

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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.

From silk fibroin peculiar characteristics, some

unconventional methods for scaffolds fabrication.

Prof. Devid Maniglio

1

BIOtech - Center for Biomedical Technologies Department of Industrial EngineeringUniversity of Trento

[email protected]

1


Silk Fibroin

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Silk Fibroin is a protein constituted on the core aminoacids sequence

–Gly–Ala–Gly–Ala–Gly–Ser–responsible of its structural assembly. The combination with several other aminoacids, determines FS numerous properties

biocompatibility, biodegradability and excellent mechanical (stiffness and toughness), optical and electronic behavior

But also different inter/intra molecular interactions driven by hydrophobic interactions, el. charge, acid/base, H-bond

D. Maniglio et al., 2010. C. Vepari et al., 2007. J. G. Hardy et al. 2008.

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2


Processing fibers to make a new material

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Scaffoldor lyophilized forstorage

To realize a new material from silk fibers you need to pass through a regeneration process: it means you have to dissolve fibers into

solution, disgragating H-bonds keeping it in shape, and re-constituting them in a different way

3


Valentina Atanasio – Final Thesis – Trento,18 july 2012

Silk fibroin


C.Vepari et al., 2007.

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3


Different perspectiveFrom the point of view of material science SF can be seen alternatively as:

A polyampholyte

likewise other proteins SF is constituted of + and - charged aminoacids

An polyamphiphile

SF is rich of hydrophobic and hydrophilic aminoacids, responsable of conformal and spatial assembly.

A polyamide

In proteins aminoacids are bond through peptide bonds that, from a chemical POV are amide bonds (-C(=O)-NH-) so SF is a polyamide and could be handled ad a thermoplastic.

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Silk Fibroin Scaffolds for Tissue Engineering fabricated by Electrodeposition

In collaboration with:Bonani W, Motta A, Migliaresi C, E. Servoli,V. Atanasio

SF as a polyampholyte

Maniglio, Devid, et al. "Electrodeposition of silk fibroin on metal substrates." Journal of Bioactive and Compatible Polymers 25.5 (2010): 441-454.

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4


Together with most of the natural polymers Silk Fibroin in water solution usually exhibits surface charges (polyampholyte).

Electric properties

Silk Fibroin can be forced to migrate in side the solution by applying an external electric field (electrophoresis)

Ē

F= (Σi qi) E – ρv

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Deposition

Electrophoretic DepositionParticle solution or suspension (colloidal) Movement of charged particle to the electrode under electric field

Electrodes don’t take part to the process

The composition of the coating is controlled by the starting solution composition

Coating :- obtained by reduction of stability of

the suspension (pH, neutralization of the mol. charge) and by instauration of intermolecular interactions

- thick coatings are possible

In particular conditions a deposition process can occur at one of the electrodesfollowing anelectrophoretic path

Loca

l dec

reas

e of

pH

9

5


Key features of the electrodeposition process

• Substrates of any shape and any size• Conformal coating or impregnation• Production of both very thin or thick coatings in form of

gels or sponges• Fast and highly efficient• Easily tunable by acting on the process variables

(voltage, concentration, volume, pH, metal surface, electrodes distance, other ions in solution…)

Based on these characteristics, electrodeposition could be an interesting technique for fibroin scaffolds preparation effective for tissue engineering

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Power generator Current acq On/off

Experimental Setup

Righello?

e-gel after deposition

e-sponge after freeze dry

Deposition chamber

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6


top skin

SEM characterization

Water electrolysis induced porosity+

freeze drying inducedsecond order lamellar porosity

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Tuning Process variablesCurrent density vs time

Deposit growth vs time (ΔV= 40 V)

Deposit growth vs pH (t=60s ΔV= 40V)

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7


TE applications

target Bone Small vessels

CELL

S

MG63(Osteosarcoma Cell line)

Osteointegration, Bone TE

MRC5(Fibroblasts Cells line)

Tunica media reconstruction

SCAF

FOLD

STarget material:

Silk fibroin spongeElectrodeposition + freeze dry

Reference material:Silk fibroin sponge

salt leaching + freeze dry

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MG63 morphology

1 daysElectrodeposited Salt leaching

alive/dead

shape

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8


1 days5 days

Electrodeposited Salt leaching

alive/dead

shape

MG63 morphology

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1 days5 days

12 days

Electrodeposited Salt leaching

MG63 morphology

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9


day

MG63 viability and proliferation

day

Cel

l num

ber

Cell Number

& ./&

6.14.2. Risultati:

I risultati ottenuti sono espressi in grafici a istogramma e a linee:

Salt leaching

E-sponge

Viability

& .+&

6.13.2. Risultati

I risultati ottenuti sono espressi in dei grafici a istogramma e a linee:

& ./&

6.14.2. Risultati:

I risultati ottenuti sono espressi in grafici a istogramma e a linee:

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Conclusions

• Electrodeposition of silk fibroin has been studied in term of the main process variables

• Sponges obtained by electrodeposition where tested in biological environment and compared with those obtained by salt leaching, generally showing faster and better cell adhesion and similar long term proliferation

• Potentialities of using electrodeposited Silk Fibroin for bone tissue (or tunica media) tissue engineering has been discussed (work still in progress)

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Potentiality of N2O foaming for the fabrication of silk fibroin scaffolds for tissue engineering

Maniglio D, Bonani W, Motta A, Migliaresi C

SF as an amphiphile

Maniglio, Devid, et al. "Silk fibroin porous scaffolds by N2O foaming." Journal of Biomaterials science, Polymer edition 29.5 (2018): 491-506.

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Valentina Atanasio – Final Thesis – Trento,18 july 2012

Silk fibroin ( Bombyx mori cocoons)


C.Vepari et al., 2007.

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Porous scaffolds

Porosity plays a crucial role for a scaffold to act as template for

tissue growth

… in fact:• porosity favours cell proliferation inside the scaffold• adequate pores volume is necessary to host a properamount of cells• open porosity is needed for the diffusion ofnutrients/metabolites/oxygen and for vascularization

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Processing polymers to get porosity

• Phase separation• Fiber bonding• Solvent casting and particulate leaching• Freeze drying (+ emulsification)• Supercritical CO2• Rapid prototyping / solid freeform fabrication

😕 low/difficult to control pore size

😕 need to remove porogens

😕 high T and P, pH drift

😕 closed porosity

😕 use of solvents

😕 compatibility with silk fibroin

Possible issues:

The introduction of new techniques can be important to open other possibilities for scaffold design.

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Using Nitrous Oxide (N2O) for foaming polymers normally used for TE, particularly in those cases where it is necessary or useful to start from a water dispersion (i.e. water proteins solutions).

A different approach

Nitrous Oxide has a good solubility in water (1.5 g/L at 15 ℃) but evenhigher in lipids and hydrophobic macromolecules (2.3 times, 20 timeshigher than N2 ) ⇒ good affinity with molecules in water havingcombined hydrophobic/hydrophilic behavior.

N2ON2O

hydrophilichydrophobic

Maniglio D, Bonani W, Migliaresi C, Motta A(2018) Silk fibroin porous scaffolds by N2O foaming, Journal of Biomaterials Science, Polymer Edition, 29:5, 491-506

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Some other advantages

• Use of water solutions at room temperature• Low pressures are required (particularly if compared with

scCO2)• No interaction with solvent (≠ CO2) ⇒ no denaturation or

precipitation• N2O has long been used as a general anesthetic and as

carrier for general anesthetic drugs• It is not flammable nor toxic• It has been reported to have bacteriostatic properties (shelf

storage)

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13


Freeze-dry foam

Sampling Stabilizationwith methanol

Sterilization Seed with cells

The foaming process

Load reactorwith FN solution

Pressurization5.5 bar (Low P)11 bar (High P)

Gas dissolution

Foaming

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Silk Fibroin foams

- Higher P ⇒ higher dispersion in the pore size

- Lower [c] ⇒ higher porosity- Presence of a small polymer lamina in the spherical pores walls- Pore throats are present

Exposure to methanol solution to induce stability in water of the foams causes the rupture of the thin walls of the pores

2%

5%

Low P High PPorosity size range

5% Low P 100-300 μm5% High P 100-400 μm2% Low P 100-300 μm2% High P 100-600 μm

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FTIR characterization

Infrared spectra of freeze-dried fibroin foams (5% High P)

Peak fitting of amide I FTIR spectra on freeze-dried fibroin foams compared with non-foamed freeze dried fibroin solution.

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Incorporation of additives

Fibroin + Gelatin Fibroin+ Gelatin+ nano-HA

The addition of Gelatin to fibroin solution (20% of FN weight):- improves foam stability- acts as emulsifier, allowing easy incorporation

of nano-microparticles, e.g., HA

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Biological tests*

DNA Content

3d 7d 14d

0

50

100

150 FN/GAFN/GA+HA

DN

A co

nten

t [µg

/ml]

LDH

3d 7d 14d

-0.5

0.0

0.5

1.0 FN/GAFN/GA+HA

c(LDH)[U/l/min] Ø LDH test shows no cytotoxicity on both

samplesØ MTT reveals a stronger activity of HA

loaded scaffold at 14dd, even with a small decrease in cell number (DNA content)

*Collaboration with prof. van Griensven, S. Schneider, E. Rosado Balmayor, Tech. Univ. of Munich

MTT

Adipose derived stem cells

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Confocal MicroscopyFN scaffolds7th day

Cell nuclei + scaffold stainingCytoscheletonCollagen I

14th day

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Confocal MicroscopyFN+HA scaffolds7th day

Cell nuclei + scaffold stainingCytoscheletonCollagen I

14th day

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Interesting effects

Wavenumber cm-1

When foaming is made extruding through a thin needle (ø 2 mm) at High Pfibroin molecules assembly is altered, due to the high shear

⇧ helix-like and β-turns structures

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The use of a small needle

silk fibroin foams obtained by extrusion through small diameter needle are stable in water

a) 5% / High P b) detail of the alignment of the

fibrous structure derived from shear stresses generated by extrusion through the thin needle

c) 2% / High P d) Evidence of the fibrillar

structure after extrusion through the thin needle at low concentration (2%)

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Foam injection

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• This technique permits the foaming of aqueous solutions macromolecules (with amphiphilic character) in a fast and repeatable way, with no need for porogen removal

• Easily tunable by acting on the process variables (N2O pressure, extrusion needle/nozzle, temperature, …?)

• Easy incorporation of additives • SF scaffolds obtained with this technique

showed adequate interaction with cellsAnd more:• It is compatible with in situ injection

(e.g. cavity filling)• Can be applied to other protein solutions

Conclusions

Based on these characteristics, N2O gas foaming can represent an interesting technique for fibroin and other polymers scaffold preparation for TERM applications.

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A low-temperature, high-pressure sintering procedure for the rapid fabrication of biosubstrates

starting from dry silk fibroin.

*Department of Chemical and Life Science, VCU, Richmond, USA

A Bucciarelli, Motta A, Quaranta A, Chiera S,Yadavalli V*

SF as a thermoplastic polymer

Bucciarelli, Alessio, et al. "A Thermal-Reflow-Based Low-Temperature, High-Pressure Sintering of Lyophilized Silk Fibroin for the Fast Fabrication of Biosubstrates." Advanced Functional Materials 29.42 (2019): 1901134.

64


Silk Fibroin

Rockwood, Danielle N., et al. "Materials fabrication from Bombyx mori silk fibroin." Nature protocols 6.10 (2011): 1612.

??????

large solid fibroin

monoliths?

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Some previous trials

3

Functional silk-based bulk materialsBenedetto Marelli, Nereus Patel, Thomas Duggan, Giovanni Perotto, Elijah Shirman, ChunmeiLi, David L. Kaplan, Fiorenzo G. OmenettoProceedings of the National Academy of Sciences Dec 2016, 201612063

Shape deformation due to shrinkage

(H2O)Fabrication time

(days/weeks)

66


An easier way?

Is it possible to treat Silk Fibroin likewise other thermoplastic polymers?

Can compression molding technique be adapted to SF manufacturing

Low process time (minutes or hours instead of weeks) facilitate translation to industry.

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The fabrication hypothesis

Preparation of a regenerated silk

fibroin water solution

Rapid cooling using liquid nitrogen and lyophilization

Pre compression stage to uniform

the material

Compression at high P and low T

69


…with the addition of a key step

Preparation of a regenerated silk

fibroin water solution

Rapid cooling using liquid nitrogen and lyophilization

Water addition via moisture absorption

Pre compression stage to uniform

the material

Compression at high P and low T

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DOE Model: studied variables

3rd year admission exam

optical properties change: from white reflective to a yellow

transparent material.

mechanical properties change:from a soft to an hard material.

Factors

A B C D

Ramp timeMax

PressureMaint. time Added water %

YieldsTransparency Stiffness400÷800 nm

Vol. normalized

Young Modulus

Physical propertiesusable as material

transformation indicators:

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Model equation

Variable +1 level -1 leveltramp (s) 600 120

Pmax (MPa) 400 200tmaint (s) 1200 0

m%W (% w/w) 20 0𝑌 = 𝑐! ∗ 𝑡"#$% + 𝑐& ∗ 𝑃$#' + 𝑐( ∗ 𝑡$#)*+ + 𝑐, ∗ 𝑚%. +𝑐/ ∗ 𝑡"#$% ∗ 𝑃$#' + 𝑐0 ∗ 𝑡"#$% ∗ 𝑡$#)*+ + 𝑐1 ∗ 𝑡"#$% ∗ 𝑚%. + 𝑐2 ∗ 𝑃$#'∗ 𝑡$#)*+ + 𝑐3 ∗ 𝑃$#' ∗ 𝑚%. + 𝑐!4 ∗ 𝑡$#)*+ ∗ 𝑚%. +𝑐!! ∗ 𝑡"#$% ∗ 𝑃$#' ∗ 𝑡$#)*+ + 𝑐!& ∗ 𝑡"#$% ∗ 𝑃$#' ∗ 𝑚%. + 𝑐!( ∗ 𝑡"#$%∗ 𝑡$#)*+ ∗ 𝑚%. + 𝑐!, ∗ 𝑃$#' ∗ 𝑡$#)*+ ∗ 𝑚%. +𝑐!/ ∗ 𝑡"#$% ∗ 𝑃$#' ∗ 𝑡$#)*+ ∗ 𝑚%.

first order terms and higher order “mixed” terms.

1 factor2 factors3 factors4 factors

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Transparency

73


Elastic modulus

74

24


Optomechanical optimization

Factors

Ramp timeMax

PressureMaint. time

Added water

%

600 s 400 Mpa 1200 s 20%

1120 ± 130 MPa (dried) to 205 ± 130 MPa (wet)

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Secondary structure

3rd year admission exam

After 12 h water exposure -> β-sheet -> no sintering

2 3 6

Random

Alpha

Turns

β-native

⥣ β⥮ β

A low initial crystallinity is important for the process to occur at low temperature.

76

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AdMSCs were seeded on LTS fibroin cylindrical samples Equivalent Poly-ϵ-caprolactone cylinder used for comparison

Cell adhesion, morphology and distribution analyzed by confocal microscopy

Cell response

actin filaments

cell nuclei

fibroin non specific absorption

Scale bar are 1000 µm for column 1, 500 µm for column 2, and 100 µm for column 3LTS: low temp sint SF PCL: Polycaprolactone as control materialScale bars are 1000 μm for column 1, 500 μm for column 2, and 100 μm for column 3.

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Conclusions

It is possible to process SF as a thermoplastic material, realizied through a fast, low-temperature compression in mold and obtaining a solid fibroin, reporting, for the first time, a thermal reflow at 40 °C for lyophilized silk fibroin.

Absorbed water plays a key role of the absorbed water vapourduring the compression phase and the fact the plasticizing effect is in competition with the self re-organization of silk fibroin secondary structure.

Large-scale solid fibroin objects produced with this method could find application in implantable bio-resorbable devices.

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26


Synthesis

From the point of view of material science SF can be seen alternatively as:

Silk fibroin peculiar characteristics, deriving from its aminoacid composition, can be used to drive protein assembly and scaffold fabrication.

The polyampholyte character can be used to realize hydrogels

The amphiphilic character can be used to realize foams

The thermoplastic character can be used to realize compact monoliths

These characters can be exploited to produce fibroin based materials with specific «flavour» usable for different biorelated applications

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1

POLYMER PROCESSING

Nuno M. Neves1,2,3

13B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine,

AvePark, 4806-909 Taipas, Guimarães, Portugal www.3bs.uminho.pt , [email protected]

2ICVS/3B’s, PT Government Associate Laboratory, Braga/Guimarães, Portugal3The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho,

Avepark, 4805-017 Barco, Guimarães, Portugal

1

BiomaterialsBiodegradables

BiomimeticsFurther information – www.3bs.uminho.pt

ICVS/3B’s, PT Government Associate Laboratory

2

MOTIVATION

90-95% of replacements are successful up to 10 years. By 10 years, 25% of all artificial joints will look loose on an X-ray. 5-10% of these will be painful and require revision.

3

LIMITATIONS

Prostheses

Ø Inflammation

Ø Aseptic loosening

Ø Revision

Bone Grafts

Ø Limited Supply

Ø Pain

Ø Donor Site Morbidity

Ø Infection (allogenous)

www.nlm.nih.gov/

4

http://www.3bs.uminho.pt/

mailto:[email protected]

2

Autologous advanced tissue engineering

therapies

5

NATURAL BASED POLYMERS

OPTIMAL BALANCE BETWEEN BIOLOGICAL PROPERTIES AND PROCESSABILITY

Natural polymers

Starch Chitosan Polyesters

PCLPLAPBS…

Synthetic polymers

MELT PROCESSABLE

6

PERFORMANCE BY DESIGN

Casanova M+, Mat. Horizons, 2020

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PROCESSING METHODS

ü Compression mouldingü Fiber meshesü Electrospinningü Solvent casting - particulate leachingü Micro-wave baking and expansionü Membrane laminationü Phase separation/inversionü High pressure based methodsü Advanced textile technologiesü Rapid prototyping technologies

8

3

SCAFFOLDS

Correlo, V, J. Biom. Mat. Res. A, 2009;Alves da Silva, Acta Biomaterialia, 2010.

Alves da Silva, J. Tis. Eng. Reg. Med., 2011;Oliveira JT, Biomat Sci. Polym. Ed., 2011;

Costa-Pinto AR, Biomacromolecules; 2009;Costa-Pinto AR, J. Tis. Eng. Reg. Med; 2012;

Costa-Pinto AR, J. Bioact. Comp. Polym., 2014

Martins A, ..., Neves NM; J. Tis. Eng. Reg. Med., 2009;

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CAD SOLID MODEL

Circles Sinusoidal Orthogonal

Fonseca D+, Biomaterials Science, 2018

10

NON-ORTHOGONAL SCAFFOLDS


Isom

etric

Top


11

SEM ANALYSIS



12

4

CHITIN / CHITOSAN

Chitin is insoluble in most common solvents, but can be converted tochitosan which is soluble in diluted acids and possesses uniqueproperties that make it suitable to be used as a biomaterial.

OCH2OH

HH

H

OH

NHCOCH3

HH

O

OCH2OH

HH

H

OH

NHCOCH3

HH

O

nGlcNAc

OCH2OH

HH

H

OH

NH2

HH

O

OCH2OH

HH

H

OH

NH2

HH

O

n

CHITIN

Deacetylation

CHITOSAN

GlcNAc

+CH3COONaNaOH, temperature

13

A twin screw extruder was used to produce new particulatecomposites of:

ü Poly(butylene succinate adipate) (PBSA)ü Poly(butylene terepthalate adipate) (PBTA)ü Poly-e-caprolactone (PCL)ü Poly(lactic acid) (PLA)

ü Poly(butylene succinate) (PBS)

Chitosan

+Biodegradable aliphatic polyesters

CHITOSAN-BASED BIOMATERIALS

MELT PROCESSABLECorrelo V+, J. Biom. Mat. Res. A, 2009.

14

CHITOSAN PARTICLES

15

STAINED SURFACES

Top surface a) 25% Ch/75% PBS; b) 70% Ch/30% PBS

Cross section c) 25%Ch/75% PBS; d) 70% Ch/30% PBS

c)

a) b)

d)

1mm

16

5

ETCHED SURFACES

Top surface of 50% Ch/50% PBS etched for 1 day in acetic acid

Before etching

17

STAINED AND ETCHED1mm

1 mm

1 mm

50C / 50PBTA samples after staining with eosin a) cross section and c) top surface50C / 50PBTA samples after etching b) cross section and d) top surface

1mm

a) b)

c) d)

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MECHANICAL PROPERTIES

0,0

0,4

0,8

1,2

1,6

2,0

PBS

PBS+30HA

25C+75P

BS

70 (2

5C+7

5PBS)+30

HA

50C+50P

BS

90 (5

0C+5

0PBS)+10

HA

80 (5

0C+5

0PBS)+20

HA

70 (5

0C+5

0PBS)+30

HA

70C+30P

BS

Esec

1%(G

Pa)

19

LOSS OF PROPERTIES

20

6

25% Ch/75% PBS – 60% SALT 25% Ch/75% PBS – 80% SALT

SCAFFOLD MORPHOLOGY - SEM

21

MICRO CT ANALYSIS

22

0

40

80

120

160

25C-75PBS-(t

hick)-60

25C-75PBS -(t

hick)-80

25C-75PBS-(t

hin )-60

25C-75PBS-(t

hin )-80

50C-50PBS -(t

hick)-60

50C-50PBS -(t

hick)-80

50C-50PBS-(t

hin )-60

50C-50PBS-(t

hin )-80

50C-50PCL-(th

ick)-60

50C-50PCL-(th

ick)-80

50C-50PCL-(th

in)-60

50C-50PCL-(th

in)-80

E se

c1%

(MPa

)

COMPRESSION MODULUS

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MELT SPINNING/FIBER BONDING

500 μm

Ø Melt Spinning

Ø Fiber Bonding*

• Average fiber diameter: 480µm

Individual Fibres

3D fiber mesh scaffold

Temperature: 150ºC

Pressure+

500 μm

24

7

FIBRE MORPHOLOGY

25%C-75%PBS

50%C-50%PBS

OPTICAL MICROSCOPY EOSIN STAINED CROSS-SECTIONS (MAGN. 120x)

25

FIBRE MESHESSEM MICROSCOPY FIBRE MESHES

26

MICRO CT ANALYSIS*

*Performed at ETH Zurich – Ralph Müller

27

Collector

High Voltage PowerSupply

Solution

Syringe

Pump

Distance

ELECTROSPINNING

28

8

ELECTROSPINNING MESHES

29

CELLS AND PATTERNS

L929 FIBROBLASTS - 1 DAY OF CULTURE

30

CELLS AND PATTERNS

31

BOVINE ARTICULAR CHONDROCYTES IN

MICROFIBER MESH SCAFFOLDS

Oliveira JT+, Tissue Engineering, 2008Oliveira JT+, J. Biomat Sci. Polym. Ed., 2011

32

9

WEEK 2

WEEK 6WEEK 4

SEM ANALYSIS

33

WEEK 2

WEEK 6

WEEK 3

WEEK 4

100x

40x40x

40x

H&E STAINING

34

WEEK 6

100x

200x

H&E STAINING

35

WEEK 2

WEEK 6

WEEK 3

WEEK 4

100x

40x

40x

40x

TOLUIDINE BLUE STAINING

36

10

WEEK 6

100x

200x

TOLUIDINE BLUE STAINING

37

WEEK 2

WEEK 6

WEEK 3

WEEK 4

40x

40x

40x

100x

ALCIAN BLUE STAINING

38

100x

200x

WEEK 6

ALCIAN BLUE STAINING

39

WEEK 2 WEEK 6WEEK 4WEEK 3

COL I

NGS

COL II

40x

COLLAGEN I & II EXPRESSION

40

11

COLL I

NGS

COLL II

100x 200xWEEK 6

COLLAGEN I & II

41

MACROSCOPIC VIEW

Oliveira JT+, J. Biomat Sci. Polym. Ed., 2011

42

CARTILAGE DEFECT IN A SHEEP MODEL

Mrugala D+, Annals Rheum. Diseases, 2008.

43

CARTILAGE REPAIR SHEEP MODEL

University Hospital Montpellier Unité Clinique, Thérapeutique Immuno Rheumatologie

44

12

9 WEEKS IMPLANTATION SHEEP MODELGross

appearanceSafranin O staining

proteoglycans

Original magnification x50

Control defects filled with fibrin glue

Chitosan powder and fibrin glue

oMSC in fibrin glue

Chitosan and oMSC in fibrin glue

Chitosan, oMSC, TGFβ3 and fibrin glue

Chitosan powder and TGFβ3

45

9 WEEKS IMPLANTATION SHEEP

Original magnification x50

Anti-type II collagen Anti-aggrecan

fibrin glue

Chitosan, MSC, TGFβ3 and fibrin

glue

chitosan powder and TGFβ3

Mrugala D+, Annals Rheum. Disease,; 2008.

46

CHONDROGENIC DIFFERENTIATION OF ADULT

STEM CELLS IN 3D CULTURES WITH ARTICULAR CHONDROCYTES

Alves da Silva ML+, JTERM, 2015

47

HUMAN CO-CULTURE MODELS

Static culture during 4 weeks

Cell source

Bone marrow

Articular cartilage

Umbilical cord –Wharton´s Jelly

Dynamic cell seeding during 24

hours

CPBS fiber meshes

Co-cultures

Direct co-culture

hACs

+

hBMSCs

hACs

+

hWJSCs

Indirect co-culture using conditioned

medium from hACs

hBMSCs

hWJSCs

48

13

GLYCOSAMINOGLYCANS

CO-CULTURES WITH hBMSCs

CO-CULTURES WITH hWJSCs

49

GENE EXPRESSION hBMSCs CO-CULTURESAGGRECAN SOX9

COLLAGEN I COLLAGEN II

50

AGGRECAN SOX9

COLLAGEN I COLLAGEN II

GENE EXPRESSION hWJSCs CO-CULTURES

51

CARTILAGE ECM FORMATION

DIRECT CO-CULTURES WITH hBMSCs

SAFRANIN O

100 µm 100 µm 100 µm 100 µm

100 µm 100 µm 100 µm 100 µm

INDIRECT CO-CULTURES WITH hBMSCs

DIRECT CO-CULTURES WITH hWJSCs

INDIRECT CO-CULTURES WITH hWJSCs

TOLUIDINE BLUE

Alves da Silva ML+, JTERM 2015

52

14

CHONDROGENESIS-INDUCTIVE NANOFIBROUS SUBSTRATE USING BOTH BIOLOGICAL

FLUIDS AND MESENCHYMAL STEM CELLS FROM AN AUTOLOGOUS SOURCE

Casanova M+, Mat Sci Eng C, 2019

53

Aminolysis1M HMD, 1h,37ºC

UV – O( 4min, each side)

NH2NH2

NH2

NFM

act

ivat

ion/

Fu

nctio

naliz

atio

n

BIOFUNCTIONALIZATION IMPLEMENTATION

NH2

Ant

ibod

y

Imm

obili

zatio

nG

row

th F

acto

rs

(GFs

) Bin

ding

EDC/NHS15 min, RT

COOH

HNNH2 HN

HN

NH2

C=H C=H C=H Blocking

3% BSA, 1h, RT HN NH2 HNHNNH2

C=H C=H C=H

Platelet Lysate (PL)

Recombinant Protein (rGF)

Selective binding of GFs

1h, RTHN

NH2 HNHN

NH2

C=H C=H C=H

COOH

NH2

NH2NH2

NH2NH2

NH2

NH2NH2

NH2

or

54

Culture28 Days

SeedhBMSCs

EXPERIMENTAL VALIDATION

NFM

Posi

tive

Con

trol

Chondrogenic MediumTGF-β3 & IGF-I

anti-TGF-β3anti-IGF-I

NFM _ IGF-1 + TGF-β3PL or recombinant GFNFM _ IGF-1

NFM _ TGF-β3Nan

ofib

rous

Syst

ems

Basal Medium

55

Maximum immobilization capacity of single antibodies at the nanofibrous substrate:

• Anti-TGF-b3 (4 µg/mL)

• Anti-IGF-I (4 µg/mL)

QUANTIFICATION OF IMMOBIL. ANTIBODY

56

15

Mixed fashion at 0.4 µg/mL anti-TGF-b3 : 3.6 µg/mL anti-IGF-I

MIXED ANTIBODIES IMMOBILIZED

Anti-

TGF-b 3

Anti-

IGF-

I

Mer

ged

View

Nega

tive

Cont

rol

50 µm

50 µm 50 µm

50 µm

57

PL GROWTH FACTORS

Donor 1 Donor 2 Donor 3 Pool

TGF-b 3 [PL] (ng/mL) 0.18 ± 0.04 0.68 ± 0.04 0.10 ± 0.08 0.27 ± 0.03

% binding 94.5 ±1 .1 98.8 ± 0.1 95.1 ± 1.0 99.3 ± 0.4

IGF-

I [PL] (ng/mL) 1.66 ± 0.13 11.31 ± 4.28 4.62 ± 2.09 6.86 ± 1.09

% binding 51.8 ± 19.3 68.9 ± 4.9 64.6 ± 5.8 77.5 ±2. 4

58

BIOLOGICAL ACTIVITY

59

ALCIAN BLUE STAINING: 28 DaysrGF PLChondro Medium

IGF-I

TGF- b3

IGF-I&

TGF- b3

200 µm 200 µm 200 µm

200 µm

200 µm

200 µm

200 µm 200 µm 200 µm

200 µm

200 µm200 µm

200 µm

60

16

CHONDROGENIC GENES

The expression was normalized against the GAPDH gene

61

IMMUNOLOCALIZATION OF COLL II: 28 DaysrGF PLChondro Medium

IGF-I

TGF- b3

IGF-I&

TGF-

b3

200 µm200 µm200 µm 200 µm

200 µm 200 µm

200 µm200 µm

Casanova M+, Mat SciEng C, 2019

62

THE INFLUENCE OF BOUND FIBRONECTIN OVER THE

CHONDROGENIC DIFFERENTIATION

Casanova M+, Biomacromolecules, 2020

63

MATERIALS AND METHODS

Basal Conditions

hBM-MSCs Differentiation

NFM _ FN+ hBMSCs

NFM _ FN

Seed

hBMSCs 28 Days

Cell ViabilityCell Proliferation

Total Protein SynthesisRT-PCR QuantificationImmunohistochemistryBiofunctional

System

Immobilization of endogenous Fibronectin

NH NH2 NHNH NHC=O C=O C=OC=O

NFM activation/ Functionalization

FN Antibody Immobilization Selective binding of FN

Anti-FN Platelet Rich Plasma (PRP)

NHNH NH2C=OC=O

NH2NH2NH2

NH2

NH2NH2

NH2

NH2NH2

NH2NH2 NH2

NH2

64

17

ANTI-FN IMMOBILIZATION CAPACITY

Secondary antibody

Primary Antibody

Alexa Fluor® 488

FN-Antibody

NHNH NH2C=OC=O

NHNH NH2C=OC=O

65

ENDOGENOUS FIBRONECTIN BINDING CAPACITY

Donor 1 Donor 2 Donor 3 Pool*

[FN] (µg/mL) 246 ± 15 111 ± 18 398 ± 28 194 ± 14* Pool of six independent donorsPRP

66

BIOCHEMICAL PERFORMANCE OF hBM-MSCS

Cell Viability Cell Proliferation

Total Protein Synthesis

67

GLYCOSAMINOGLYCANS DEPOSITION

68

18

ALCIAN BLUE STAINING AND CELL MORPHOLOGY

hBMSCs cultured during 28 days

FIBRONECTINBasal Medium Chondrogenic Medium Soluble Bound

69

CHONDROGENIC TRANSCRIPTS EXPRESSION

COMPSox 9

Aggrecan Collagen Type II Collagen Type X

Collagen Type Ia

70

IMMUNOEXPRESSION OF MATRIX PROTEINShBMSCs cultured during 28 days

Collagen Type II

Collagen Type IaFIBRONECTIN

Basal Medium Chondrogenic Medium Soluble Bound

FIBRONECTINBasal Medium Chondrogenic Medium Soluble Bound

Casanova M+, Biomacromolecules, 2020

71

SUMMARY

The surface functionalized scaffolds support primary adult stem cell viability, expansion and differentiation ex-vivo and the antibody immobilization strategy may be used for other specific applications

72

19

TEAM

73

FUNDING

Cells4_IDs - PTDC/BTM-SAL/28882/2017

FRONTHERA - NORTE-01-0145-FEDER-0000232

FCT PHD PATH - PD/00169/2013

74

ESAO-TERMIS Winter School (Online)

75

Juthamas RATANAVARAPORN

Siriporn DAMRONGSAKKUL

Sorada KANOKPANONT

Biomedical Engineering Program

Faculty of Engineering

Chulalongkorn University

Controlled Release Technology for Curcumin and Its

Synergistic Compounds for Treatment of Prevalent Diseases

REMIX online Seminar 2021

Tissue Engineering and Controlled Release Applications of Thai Silk Fibroin-based Materials

Cancer

VascularWound dressing, skin substitute3D bioprinting

Bone regenerationOsteoarthritis

Curcumin (1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a major

component of the perennial herb Curcuma longa (turmeric) classified as a polyphenol

hydrophobic molecule.

Various pharmacological activities of curcumin

Curcumin is one of the most ancient medicinal herbs and is being used for the treatment

of various health problems for a thousand hundreds of years in Asian medicine.

• anti-inflammatory

• anti-oxidant

• anti-cancer

• anti-microbial

• immunomodulatory

• cardio-protective

• nephro-protective

• hepato-protective

• anti-neoplastic

• anti-rheumatic

• hypoglycaemic effects

Curcumin and prevalent diseases

4

Limitations of curcumin

• Water insolubility

• Poor systemic bioavailability

• Low stability at physiological pH due to its rapid

metabolism and short half-life

Possible solutions

• Controlled release system

• Combination with synergistic bioactive compounds

Spongy scaffold

Microspheres

Film/membraneHydrogel

Nanofiber mat

Controlled release carriers

Injectable Implantable

Micelle

Synergistic Combinations of Curcumin

M.S. Hosseini-Zare et al. European Journal of Medicinal Chemistry 210 (2021) 113072

The gelatin/silk fibroin microspheres were

fabricated by w/o emulsion and glutaraldehyde

crosslinking techniques.The microspheres with size 200 μm were

collected by sieve.

Curcumin and/or

piperine were

loaded on the

microspheres.

Crosslinking percentage of G/SF microspheres, analyzed by TNBS

In vitro degradation profile of G/SF microspheres in collagenase enzyme

50/50, 30/70

70/30

100/0

The microspheres with SF more than 50% showed high percentage of

crosslinking and were not degraded in collagenase solution.

Encapsulation and loading efficiency of curcumin and piperine

Either curcumin or piperine can be successfully encapsulated in all microspheres, however,

the encapsulation efficiency in the dual system was lower than that of microspheres loaded

with curcumin or piperine alone

In vitro release profile of curcumin and piperine from G/SF microspheres

Slow degrading microsphere (high SF content) released

curcumin and piperine at slower rate.

Thai silk fibroin-based microspheres for the treatment of osteoarthritis

Osteoarthritis (OA) is an inflammatory joint disease mostly found in elderly

population around the world and significantly affects on their quality of life. OA is

characterized by a progressive disintegration of articular cartilage, overgrowth of

subchondral bone, and inflammation in synovial membrane.

Consequently, these conditions result in loss of

joint function, disability and chronic pain.

Current treatments of OA• Non-steroidal anti-inflammatory drugs

(NSAIDs) >> side effects such as

cardiovascular toxicity

• Intra-articular therapy e.g. glucocorticoids and

hyaluronic acid >> controversial results

The reduction of inflammation in OA joint is

necessary and can subsequently delay the

progression of OA, and improve joint

mobility thereafter.www.artritereumatoide.com.br

www.zoetisus.com

Various studies reported the potential of curcumin

for the treatment of OA by

o suppress inflammation in synovial fibroblasts

o inhibited NF-κB activity in fibroblast-like

synoviocytes and chondrocytes

A safety curcumin dose of oral administration

upto 8–12 g/day

HOWEVER, curcumin has poor bioavailability when

taken as oral dietary supplement.

Int Immunopharmacol 2010;10:605–610

Int J Mol Med 2007;20:365–72

Biochem Pharmacol 2007;73:1434–45

Controlled release system

Experimental groups

Gr.1: no OA induction (normal)

Gr.2: treatment with normal saline

Gr.3: treatment with gelatin microspheres (G100) encapsulating curcumin

Gr.4: treatment with gelatin/silk fibroin (30/70) microspheres encapsulating curcumin

t = -4W

Intra-articular

inject MIA to

induce OA

t = 0W

Treatment

t = 1W

IL-6

t = 4W

IL-6

t = 8W

IL-6

x-ray

histology

The in vivo anti-inflammation efficacy of the gelatin/silk fibroin microspheres

encapsulating curcumin was investigated in monosodium iodoacetate-induced OA.

8-week-old Wistar rat

Ratanavaraporn J. et al., Inflammopharmacol (2017) 25:211–221

+

Concentration of serum IL-6 by ELISA

The IL-6 levels of OA rats treated with gelatin and

gelatin/silk fibroin (30/70) microspheres encapsulating

curcumin were significantly reduced after 1 week of

treatment.

X-ray radiographical and histological images of articular

joint of OA rats at 8 weeks after received a single treatment

In vivo osteoarthritis treatment

Grade Degree of OA Descriptions

Gr. 1 0 No No radiographic signs of OA

Gr. 2 2 Moderate Presenting with obvious sclerosis, osteophyte <0.2mm, joint effusion

Gr. 3 1 Mild Presenting of subchondral sclerosis, no osteophyte, slightly joint effusion

Gr. 4 0 No No radiographic signs of OA

• The radiographic signs of OA were not observed in the rats treated with gelatin/silk fibroin (30/70)

microspheres encapsulating curcumin at 8 weeks.

• The histological OA scores of rats treated with gelatin/silk fibroin (30/70) microspheres

encapsulating curcumin were similar to those of normal rats at 8 weeks after treatment.

Radiographic grading of articular cartilage lesion of OA rats at 8 weeks after received a single treatment

Grade

Articular joint Gr.1 Gr.2 Gr.3 Gr.4

I. Structure

0.0 5.0 7.3 2.7

II. Cell 1. Tangential zone 1.0 2.0 2.0 0.7

2. Transitional and Radial zone 3.5 7.3 8.3 4.7

IV. Tidemark

0.5 1.0 1.0 0.7

V. Pannus formation

0.0 1.7 1.3 0.3

Histologic and histochemical grading of articular joint and synovial tissue change of OA rats at 8 weeks after received a single treatment

In vivo osteoarthritis treatment

Thai silk fibroin-based microspheres for the treatment of cancer

G80SF2050.3 ± 8.5 µm 47.9 ± 5.6 µm

G50SF50

DHT-crosslinked G/SF microspheres Animal Preparation

▪ CaSki cells were inoculated in the middle area of dorsal skin-fold chamber

▪When the tumor volume was 100–120mm3, the

mice were subcutaneously injected with curcumin

absorbed microspheres for once a week

▪ The changes of tumor volume were measured

and tumor reduction rate was calculated

% Tumor reduction = 100]1[0

−TV

TVn

Where, TVn and TV0 are the final and initial tumor

volume, respectively

100 µm

Confocal images of tumor

microvasculature

Study of Tumor Microvasculature

Fluorescence images of tumor microvasculature

▪ Fluorescence FITC-

labeled dextran were

injected in the jugular

vein

▪ The tumor

microvasculature was

visualized and

neocapillary density

(NCD) was calculated

TV= 193.98 mm3

Before treatment

(Day0)

TV= 429.62 mm3

After treatment

(Day28)

%NCD = 33%

Tumor size reduction >>> 54.85%

In vivo results

Silk fibroin/gelatin sponge

Curcumin loading

SF100

Curcumin/DHA loading

DHA loading

SF80G20

SF50G50

SF20G80

SF15G85SF10G90SF5G95G100

Encapsulation and loading efficiency of curcumin and/or DHA in SF/G sponges

The high encapsulation efficiency of both curcumin and DHA were achieved.

In vitro release profiles of curcumin and/or DHA from SF/G sponges

The sustained release profiles of both curcumin and DHA were achieved.

CaSki cells cultured with sponges releasing curcumin and/or DHA

CaSki cells cultured with curcumin and/or DHA solution

In vitro anti-proliferation of cancer cell lines

The dual release of curcumin

and DHA from sponges

effectively killed cancer cells

in vitro particularly at day 1

Formation of SF/anionic surfactant hydrogel incorporating curcumin

SF solution Anionic surfactant solution

37 oC, pH 7.4

SF hydrogel

SF hydrogel

incorporating curcumin

Gelation profile of SF/SDS and SF/STS hydrogels

Gelation time of SF-based hydrogels

• SDS and STS that have long alkyl

chain lengths and high negative

charges could accelerate the gelation

of SF to occur within 14–42 min in a

concentration-dependent manner.

• SOS that has a short alkyl chain

length and low negative charge slowly

induced SF to gel at around 113–144

h.

*

Indirect cytotoxic test of SF/STS hydrogelSolubility of curcumin in anionic surfactants

SOS SDS STS

• STS could solubilize curcumin at highest

concentration because it formed mor micelle

molecules to solubilize curcumin than SDS

and SOS.

• SF hydrogel induced by STS had no

cytotoxic effect on L929 cells at all dilutions

In vivo wound healing efficiency of Thai SF hydrogels

controlled releasing curcumin

SF/STS hydrogel + curcumin

• The SF/STS hydrogels and SF/STS

hydrogels + curcumin resulted in

significantly reduced wound area since 3

days after treatment.

Number of infiltrated neutrophils in wound

tissue after treatment for 3 and 7 days

Anti-inflammation effect of Thai SF hydrogels controlled

releasing curcumin

The less number of neutrophil cells was

observed for the wounds treated with

SF/STS hydrogels and SF/STS

hydrogels + curcumin compared to

those of non-treated and fibroin gel-

treated wounds.

Re-epithelialization of wound after treatment

for 7 and 14 days

In vivo wound healing efficiency of Thai SF hydrogels

controlled releasing curcumin

The efficiency of wound healing of

SF/STS hydrogel and SF/STS

hydrogel + curcumin was comparable

to the clinically available fibrin gel.

Thank you

silkfibroin - unitrento

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