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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
1
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Silk Fibroin
2
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.
2
2
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Processing fibers to make a new material
3
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
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Valentina Atanasio – Final Thesis – Trento,18 july 2012
Silk fibroin
D. Maniglio et al., 2010. C. Vepari et al., 2007. J. G. Hardy et al. 2008.
C.Vepari et al., 2007.
4
3
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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.
5
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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.
6
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
8
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
10
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Power generator Current acq On/off
Experimental Setup
Righello?
e-gel after deposition
e-sponge after freeze dry
Deposition chamber
11
6
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
top skin
SEM characterization
Water electrolysis induced porosity+
freeze drying inducedsecond order lamellar porosity
12
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Tuning Process variablesCurrent density vs time
Deposit growth vs time (ΔV= 40 V)
Deposit growth vs pH (t=60s ΔV= 40V)
13
7
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
14
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
MG63 morphology
1 daysElectrodeposited Salt leaching
alive/dead
shape
15
8
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
1 days5 days
Electrodeposited Salt leaching
alive/dead
shape
MG63 morphology
16
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
1 days5 days
12 days
Electrodeposited Salt leaching
MG63 morphology
17
9
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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:
18
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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)
23
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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.
24
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Valentina Atanasio – Final Thesis – Trento,18 july 2012
Silk fibroin ( Bombyx mori cocoons)
D. Maniglio et al., 2010. C. Vepari et al., 2007. J. G. Hardy et al. 2008.
C.Vepari et al., 2007.
25
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
26
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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.
28
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
29
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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)
30
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
31
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
32
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
33
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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.
35
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
36
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
37
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Confocal MicroscopyFN scaffolds7th day
Cell nuclei + scaffold stainingCytoscheletonCollagen I
14th day
38
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Confocal MicroscopyFN+HA scaffolds7th day
Cell nuclei + scaffold stainingCytoscheletonCollagen I
14th day
39
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
40
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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%)
41
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Foam injection
43
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
• 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.
44
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Silk Fibroin
Rockwood, Danielle N., et al. "Materials fabrication from Bombyx mori silk fibroin." Nature protocols 6.10 (2011): 1612.
??????
large solid fibroin
monoliths?
65
20
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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.
67
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
…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
70
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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:
71
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
72
23
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Transparency
73
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
Elastic modulus
74
24
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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)
75
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
25
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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.
79
Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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.
80
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Devid Maniglio - From silk fibroin peculiar characteristics, some unconventional methods for scaffolds fabrication.
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
81
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
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
7
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;
9
CAD SOLID MODEL
Circles Sinusoidal Orthogonal
Fonseca D+, Biomaterials Science, 2018
10
NON-ORTHOGONAL SCAFFOLDS
Circles Sinusoidal Orthogonal
Isom
etric
Top
Fonseca D+, Biomaterials Science, 2018
11
SEM ANALYSIS
Circles Sinusoidal Orthogonal
Fonseca D+, Biomaterials Science, 2018
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)
18
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
23
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