Regeneração de

Tecidos
MEB 2022-2023
Ricardo Baptista
Intended learning outcomes
1. Know the principles of tissue regeneration from a biological and technological perspective
2. Know applications of regenerative medicine
3. Know laboratory techniques for cell cultures
4. Design of three-dimensional scaffolds for tissue regeneration
5. Know how to build Scaffolds using conventional techniques and additive manufacturing

a. Scaffolds design using CAD and parametric programming language

b. Scaffold production using conventional techniques
c. Solvent-casting and particle leaching
d. Production of Scaffolds by additive manufacturing

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Assessment
• Labs + Test

a) Scaffold numerical modeling and FDM

3D printing
b) Lost mold ceramic scaffolds production
for bone regeneration
c) Alginate film production for skin
regeneration
d) Hydrogel production for articular
regeneration

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References

https://doi.org/10.1016/j.polymer.2017.11.049

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References

https://doi.org/10.1016/j.oceram.2022.100219

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References

http://dx.doi.org/10.1016/j.compscitech.2016.03.013

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References

https://doi.org/10.1016/j.jddst.2023.104298

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References

http://dx.doi.org/10.1016/j.msec.2017.05.017

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Introduction
Tissue
Engineering
 • Tissue engineering is a biomedical engineering
discipline that uses a combination of cells,
engineering, materials methods, and suitable
biochemical and physicochemical factors to
restore, maintain, improve, or replace different
types of biological tissues
• The term is closely associated with applications
Tissue that repair or replace portions of or whole tissues:
• Bone,
Engineering • Cartilage,
• Blood vessels,
• Bladder,
• Skin,
• Muscle, etc.

• https://en.wikipedia.org/wiki/Tissue_engineering

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Scaffolds
• Artificial structure used to support three-Dimensional (3D) tissue
formation
• Scaffolds can be used as acellular systems or as vehicles for cells
and/or drugs
• Once implanted into the injured site, acellular materials should
allow proper host cell colonization for regeneration purposes
• Alternatively, scaffolds can be combined with different types of
cells able to promote bone formation in vivo either by
differentiating towards the osteogenic lineage or releasing
specific soluble molecules

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 • An ideal scaffold suitable for TE applications should
Scaffold allow or improve cell viability, attachment,
proliferation and homing, differentiation,
Features vascularization, host integration and, where
necessary, load bearing
• Enable easy handling without extensive
preparatory procedures in the operation theatre
and allow minimally invasive implantation
• Be sterilizable by industrial techniques and
reproducible on a large scale with cost effective
processes
• Meet the applicable Agency or competent
authority requirements

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Biological • A scaffold must be biocompatible and
nontoxic
requirements • Cells must adhere, function normally,
proliferate, differentiate and produce new
matrix
• Bioresorbable and biodegradable features
are also important in order to allow tissue
formation to occur in tandem with
degradation
• Bioactive scaffolds are designed to
promote proper cell migration or
differentiation, tissue neoformation and
integration in the host, avoiding
undesirable processes such as scarring

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Structural • Combining computer assisted design
(CAD) with computer assisted

features manufacturing (CAM), custom-made,

personalized and anatomically shaped
scaffolds can be manufactured
• Optimal pore size, ranging from 200 to
500 μm, is considered ideal for bone
regeneration and vascularization
• Scaffold mechanical properties, such as
elastic modulus, tensile strength, fracture
toughness, fatigue, and elongation
percentage, are considered crucial in TE
and should be modulated or tailored in
order to match with those found at the
site of implantation

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Composition • The majority of scaffolds that are currently
used for TE applications are polymers,
bioactive ceramics and composites
• Polymers can be natural or synthetic.
Naturally derived polymers, such as fibrin,
hyaluronic acid, chitosan and collagen exhibit
good biocompatibility; disadvantages are
represented by a degradation rate difficult to
control and low mechanical stability
• Synthetic polymers, like polyanhydride,
polypropylene fumarate (PPF),
polycaprolactone (PCL), polyphosphazene,
polylactic acid (PLA), polyether ether ketone
(PEEK) and poly(glycolic acid) (PGA) display a
controlled degradation rate, the possibility to
design or tune mechanical properties and to
fabricate complex shapes, cell attachment
improvement
http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Composition • An important class of polymers utilized in
TE is represented by hydrogels, hydrophilic
polymer networks that may absorb water
from 10 to 20% up to thousands folds
their dry weight
• This property allows cells to adhere,
proliferate, and differentiate
• Hydrogels, both naturals (agarose, alginate
and gelatines) and synthetics (poly(vinyl
alcohol)-based), are able to mimic ECM
topography and to deliver bioactive
molecules

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Composition • Bioactive ceramics can be of natural or
synthetic origin (coralline, HA, tricalcium
phosphate - TCP - sulphate, bioactive glass
- BG - and calcium silicate)
• Chemically similar to bone, they show high
compressive strength and low ductility,
thus providing high resistance to
deformation but, at the same time,
brittleness
• Composites consist of a combination of
two or more materials with different
properties, each displaying only some
advantages and specific drawbacks

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Composition • Polymer-ceramic composites are really
biomimetic, since bone is, in fact, a
composite material made of a mix of
inorganic HA crystals and organic collagen
fibers
• The incorporation of inorganic inclusions
such as bioceramic and bioglass particles,
carbon nanotubes, or magnesium metallic
or alloy particles seems to positively affect
scaffold mechanical properties

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Manufacturing • 3D fabrication available technologies can
be divided into two main categories:
technologies • Conventional
• Rapid Prototyping
• Conventional techniques use subtractive
methods in which parts of the material are
removed from an initial block to reach the
desired conformation
• A key issue is a limited ability to control
shapes and geometries or to incorporate
internal architecture or curved channels
• the use of organic solvents may
compromise cell viability or functions,
even when only residues remain

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Manufacturing • RP techniques are additive fabrication
processes (also defined as “additive
technologies manufacturing” or “solid free form
fabrication”) that manufacture the final 3D
object via deposition of overlying layers
• A more punctual control of porosity, pore
size and mechanical and chemical
properties is possible
• RP technology is based on the possibility
to obtain objects (CAM) starting from a 3D
mathematical model definition (CAD)

http://dx.doi.org/10.1016/j.msec.2017.05.017

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Solvent-casting and particle leaching
• Solvent-casting and particle leaching are techniques where a polymer solution is dissolved in
a solvent with uniformly distributed salt particles of a specific size
• The solvent evaporates leaving a salt particles-embedded matrix; this is immersed in water
where the salt leaches out to produce a highly porous structure
• Key advantages are high scaffold porosity and the feasibility to tune pore size

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Solvent-casting and particle leaching
• Disadvantages of this technique are represented by the fact that the process can only
form simple shape scaffolds (flat sheets and tubes) and that the residual solvent could be
harmful to cells

• Cao et al. scaffolds, displaying high porosity and interconnected pores, were investigated
for their ability to repair critical bone defects in rat femoral medial-epicondyles
• Quantitative image analysis and qualitative histological evaluations showed that bone
formation began within 14 days of surgery and was completed after 30 days
• By day 90 bone replacement was almost completed

https://doi.org/10.1016/j.bone.2009.09.031

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 • PGA is soluble in highly fluorinated organic solvents (HFIP)
• PGA was completely dissolved in HFIP (CO = 0.2 g/ml) at room temperature
• β-TCP powder was then uniformly mixed in the solution
• solvent was evaporated in draft equipment at room temperature for 2 days
• matrices were then immersed in distilled water for 3 days to remove the
NaCl

• average pore sizes of

PGA/β-TCP (1:1)
scaffolds and PGA/β-
TCP (1:3) scaffolds were
504.2 ± 93.5 μm and
483.3 ± 113.6 μm,
respectively
• opening porosities of
PGA/β-TCP (1:1)
scaffolds and PGA/β-
TCP (1:3) scaffolds were
93.6 ± 2.0% and
88.4 ± 0.7%,
respectively
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https://doi.org/10.1016/j.bone.2009.09.031

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 • In the PGA/β-TCP (1:1) and PGA/β-TCP (1:3) groups, the
mineralization and osteogenesis began throughout defect area at 14
days after surgery, and advanced rapidly
• Until 90 days after surgery, the defect areas showed the appearance
of natural bone and smooth bone surface shape
• Through all the progress of bone remodeling, the mineralization in
PGA/β-TCP (1:3) group was higher than in PGA/β-TCP (1:1) group

• However, in no implant control group, the mineralization and

osteogenesis began from the basal part of the defect, and grew
gradually upward to defect surface layer
• In HAP control group, the brightness variation in the HAP control
group was not obvious, due to the large remaining of HAP
• Until 90 days after surgery, the surface shape was irregular

https://doi.org/10.1016/j.bone.2009.09.031

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 Procedure
https://doi.org/10.1016/j.oceram.2021.100084

• alumina powder SG4000 (Almatis) was mixed

with a hydrophobic thermoplastic vinyl
polymer binder (4 wt%) and a suitable
alcoholic solvent system
• mixture was mechanically mixed in high
shear for 10–15 min while adding the solvent
gradually until a smooth dough was formed
• mixture with slightly lower viscosity was
formed by increasing the solvent content
and working time for adding the porogen
• NaCl particles of size 100–700 μm were
mixed in the dough

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 Results
https://doi.org/10.1016/j.oceram.2021.100084

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 • As expected the strength decreased from 54
Results MPa to 26 MPa with an increase in NaCl
content from 39 wt% to 49 wt%

https://doi.org/10.1016/j.oceram.2021.100084

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Aplication • Dental root implant analogue

https://doi.org/10.1016/j.oceram.2021.100084

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 • In the freeze drying process, also known as
lyophilization, a synthetic polymer is firstly
dissolved into a suitable solvent
• Then, the polymer solution is cooled down
below its freezing point, leading to the
solidification of the solvent that is
evaporated via sublimation, leaving a dry
Freeze drying scaffold with numerous and interconnected
pores

• Avoid high temperatures that could reduce

the activity of incorporated biological factors
• Moreover, pore size can be easily controlled
by tuning the freezing regime
http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Freeze drying

http://dx.doi.org/10.1016/j.msec.2017.05.017
http://dx.doi.org/10.1016/j.msec.2017.05.017

Freeze drying
• Chen et al. utilized freeze-dried-produced calcium-Alginate
scaffolds
• to seed human osteoblasts
• The engineered tissues were then cultured in a perfusion-
based bioreactor system, with the aim to mimic in vivo bone
environment
• Results highlighted the formation of bone-like tissues

• Disadvantages of this technique are the lengthy timescales,

high energy consumption, the use of cytotoxic solvents

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 • TIPS is a low temperatures process where a
polymer solution is quenched and undergoes
Thermally a liquid-liquid phase separation to form two
phases: one polymer-rich and the other
induced polymer-poor
• The polymer rich phase solidifies and the
phase polymer poor one is removed, leaving a
separation highly porous, nanoscale fibrous network
• Low temperatures favor the incorporation of
bioactive molecules

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Thermally
induced
phase
separation

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 • Gas foaming technique was developed to avoid
organic, cytotoxic solventes
• The process uses relatively inert gas-foaming agents
(carbon dioxide, nitrogen) to pressurize molded
biodegradable polymers with water or fluoroform,
until they are saturated and full of gas bubbles
• This technique typically produces sponge-like
Gas foaming structures with an average pore size in the range of
30–700 μm and a porosity up to 85%

• Drawbacks of gas foaming technology include the use

of excessive heat during compression molding, close,
non-interconnected pore structures and a nonporous
skin layer at the scaffold surface

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Gas foaming

http://dx.doi.org/10.1016/j.msec.2017.05.017
https://doi.org/10.1016/j.msec.2015.03.002

• 4.5 g of poly(ε-caprolactone) diol (PCL diol) were

reacted at 70 °C with selected amounts of
Gas foaming polymethylene polyphenyl isocyanate (PMDI)
under vigorous stirring in the presence of 5.8 mg of
1,4-diazabicyclooctane (DABCO, > 98%)
• To obtain foams at different stiffness levels, two
different amounts of PMDI were tested: 13 wt.%
and 19 wt.% (vs. total)
• PMDI incorporation was followed by a rapid
addition of 200 μL of a 6% w/v albumin aq.
solution
• After vigorous stirring for 1 min to start the
foaming reaction, the pre-polymer was transferred
in a polyethylene mold, where complete curing
and hardening took place in ca. 1 h

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https://doi.org/10.1016/j.msec.2015.03.002

Gas foaming

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 Powder-forming process
• The powder-forming process allows the fabrication of
porous ceramic scaffolds
• In details, a ceramic particle suspension in a suitable liquid
(such as water or ethanol) called slurry, is used to prepare
green bodies
• Among these processes, the replication technique, also
named the ‘polymer-sponge’ method, including a
crystallization phase, offers the potential of forming
uniform dispersion of ceramic powder within a template,
resulting in controllable pore size, high porosity and
interconnectivity

http://dx.doi.org/10.1016/j.msec.2017.05.017

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Powder-forming process https://doi.org/10.1016/j.jeurceramsoc.2019.10.029

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 Colloidal processing, foam replication
https://doi.org/10.1016/j.oceram.2022.100219

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 • Mechanical strength of brittle materials is strongly affected by
porosity
• Despite the superior porosity and pore sizes of the produced
Results scaffolds containing 15 wt% PVA, it achieved slightly better
compressive strength
• An indication that his structs are, in fact, denser

https://doi.org/10.1016/j.oceram.2022.100219

10% PVA 15% PVA

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 • Electrospinning utilizes electrical charges to draw,
by means of a syringe pump, fine fibers up to the
nanometer scale and create, with a collector, a
nanofibrous architecture with surface areas able
to adsorb proteins and binding sites to cell
membrane receptors
• A standard system requires four major
Electrospinning components: a spinner with a metallic needle, a
syringe pump, a high-voltage power supply, and
a grounded collector
• The electric field strength overcomes the surface
tension of the droplet and generates a charged
liquid jet that is then elongated and whipped
continuously by electrostatic repulsion until it is
deposited on the grounded collector

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Electrospinning

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 http://dx.doi.org/10.1016/j.compscitech.2016.03.013

Hydrogel

• Hydrogels based on multitudinous

polymers have been studied for
replacement or repair of damaged
articular cartilage including:
• poly(lactic-co-glycolic acid)
(PLGA),
• hyaluronic acid/poly(-
ethylene glycol) (HA/PEG),
• polyvinyl alcohol/poly(vinyl
pyrrolidone) (PVA/PVP),
• collagen
• and gelatin

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 http://dx.doi.org/10.1016/j.compscitech.2016.03.013

Procedure

• PVA composite hydrogels were prepared by a freezing-thawing

method
• The PVA solution and PEEK solution were prepared by dissolving
them in dimethyl sulfoxide at 85 C with stirring for 6 h
• PVA content was adjusted to 75 wt % of the PEEK mass
• Mixture of PVA-b-TCP can also be prepared by the same method
• Mixtures of PVA-PEEK and PVA-b-TCP were poured into a self-made
mould and with temperature set at -20 ºC, was placed in a
refrigerator for 20 h
• Then it was taken out and thawed at room temperature for 6 h
• The freezing and thawing process was repeated 9 times

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 http://dx.doi.org/10.1016/j.compscitech.2016.03.013

Results
Compression Tension

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 RP techniques

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Stereolithography

• The term SL was coined by Charles

Hull who invented the technique
described in his U.S. Patent issued
in 1986
• He defined a method and
apparatus for making solid objects
by successively printing thin layers
of an ultraviolet (UV) curable
material one on top of the other
• Schematically, a SL system consists
of a tank of photo-sensitive liquid
resin, a moveable build platform,
an UV laser to irradiate the resin
and a dynamic mirror system
http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Stereolithography
• However, the use of bioceramic scaffolds can be
problematic due to viscosity that may hamper the
process
• For this reason an indirect fabrication method has
been developed where an epoxy mold is first
created by SL and then a suspension of ceramic
acrylate is cast into the mold that is then removed
by thermal treatment

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 as-built CaP/HDDA scaffolds with different percent elongation (%EL)

Stereolithography

https://doi.org/10.1016/j.ceramint.2020.09.164
 Stereolithography

https://doi.org/10.1016/j.ceramint.2020.09.164

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Fused deposition
modeling
• In FDM process molten thermoplastic
materials, extruded from a nozzle, are
deposited onto a base platform following a
path, which is predefined by CAD and CAM
• When each layer in the xy plane is finished,
the platform (z axis) is lowered and the
procedure is repeated
• 3D scaffolds with controllable pore size and
porosity can be fabricated by changing the
material deposition amount, the spacing
between the material paths and the height
interval (z axis)
http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Fused deposition modeling

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 • The main advantages are: material high porosity
and good mechanical strength, no toxic solvent
requirement and flexibility in material handling and
Fused processing
• FDM can be also utilized to produce composites
deposition like PCL-HA or PCLTCP that are used in BTE for their
mechanical and biochemical properties
modeling
• Furthermore, its application to biodegradable
polymers, apart from PCL, may be limited

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Fused deposition modeling
https://doi.org/10.3390/ijms17040595

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 Selective • SLS, developed by the University of Texas
in Austin in 1986, binds together powder

laser particles in thin layers with a high-power

laser, such as carbon dioxide
• Subsequent formed layers are then bound
sintering to the previous ones, following the cross-
sectional information carried by the
predefined CAD data
• Because the powders are maintained with
low compaction forces after the sintering
process to form new layers, structures
have an internally porous structure
suitable for a bone scaffold

http://dx.doi.org/10.1016/j.msec.2017.05.017

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 Selective
laser
sintering

http://dx.doi.org/10.1016/j.msec.2017.05.017
 Selective laser sintering

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