Bone tissue

enineering
Regeneração de Tecidos
Ricardo Baptista 2022-23
Content
• References
• http://dx.doi.org/10.1016/j.msec.2017.05.017
• https://doi.org/10.1016/j.msec.2020.110716
• https://doi.org/10.1016/j.actbio.2018.11.039

• Bone Tissue
• Strategies for bone regeneration
• Scaffold manufacturing techniques
• Scaffold properties
• Conclusions

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Bone Tissue
• Bone loss has, in general, significant effects on patient's quality of life
• As a consequence, bone-related medical treatments and costs are increasing
• Moreover, because of a prolonged life expectancy and an aging world
population, there is a rapid increase in musculoskeletal pathologies such as:
• fractures,
• low back pain,
• scoliosis,
• osteoporosis,
• bone infection or tumors,
• congenital defects and oral and maxillofacial pathologies,
• as well as rheumatic diseases like osteoarthritis

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Bone Tissue
• Bone is made up of 2 parts: the spongy cancellous bone and the stiffer cortical
bone
• It comprises of both organic parts like the protein collagen (Type I collagen
accounts for 97 wt percent of ECM protein) and inorganic mineral phase like
hydroxyapatite which provides strength to the entire framework
• A human body has about 270 bones, some of which fuse together till the
person has about 206 bones well into adulthood

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Bone Tissue

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Bone Tissue
• The cortical bone is mainly composed of micrometer scale osteons as well as
haversian canals which fit along with blood vessels, canaliculi and other such
components in the limited porosity present within the cortical bone whereas bone
marrow is found in the cancellous bone which has up to 90% porosity
• The properties of bone are heavily dependent on their age and location inside the
body
• This is why, it is considered to be an anisotropic material
• Generally, the moduli of the bone ranges from 1 to 20 GPa

• When it comes to the cancellous or trabecular bone with about 50–90% porosity, the
maximum strength is found in the femur
• Its strength (roughly 6.8 MPa) is approximately double that found in the vertebra (2.4
MPa)

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Strategies for bone regeneration
• Bone is the most commonly transplanted tissue after blood
• The drawbacks in autograft technique (either vascularized or not), which
represents the current gold standard, are: supply limitation, variable resorption,
risk of donor site morbidity, high rate of failure in specific sites and the need of
a second surgery
• The problems associated with allografts (from human cadavers or living
donors), such as Demineralized Bone Matrix and xenografts (animal source) can
be pathogen transmission and rejection by the recipient's body

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Strategies for bone regeneration
• Metal implant strategy (titanium alloys and stainless steel) such as joint
prostheses, plates and screws, currently utilized to provide mechanical and
structural support for joint arthroplasties and long bone and vertebral fractures,
presents limitations due to non-degradability, high rigidity, fatigue, fracture, lack
of integration into the host tissue, extrusion and infection

• Scaffolds
• A scaffold can be described as an artificial structure used to support three
Dimensional (3D) tissue formation

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Strategies for bone regeneration
• 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

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Strategies for bone regeneration

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Strategies for bone regeneration
• The majority of scaffolds that are currently used for bone tissue enineering
applications are polymers, bioactive ceramics (glasses) and hybrids (composites)
• Depending on composition and intended use, they may be injectable or rigid

• Naturally derived polymers, such as fibrin, hyaluronic acid, chitosan and collagen
exhibit good biocompatibility, osteoconductivity and low immunogenicity
• 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 bone mechanical properties and to fabricate complex
shapes, cell attachment improvement (negatively-charged chemical groups) and the
potential to deliver soluble molecules

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Strategies for bone regeneration
• 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

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Scaffold manufacturing techniques

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Scaffold manufacturing techniques
• The solvent casting/particulate leaching (SCPL) is the oldest technique used for
scaffolds fabrication
• The concept of the method depends on the dispersion of porogens into a
polymer solution
• The scaffolds obtained after solidification of the polymer and dissolution of the
porogens which left a high volume of pores resulting in a porous scaffold is also
known as the foam

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Scaffold manufacturing techniques
• In the study of Cao and colleagues, 3D PGA/β-TCP
composite scaffolds were fabricated using this
method
• The 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
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Scaffold manufacturing techniques

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

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Scaffold manufacturing techniques

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Scaffold manufacturing techniques
• Melt molding technique is based on mixing of gelatin microspheres with a
specific polymer powder in a Teflon mold
• The mixture is then heated above the polymer glass transition temperature
• The best polymer for this method is known to be poly (lactic glycolic acid)
(PLGA) due to its lower glass transition temperature

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Scaffold manufacturing techniques

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Scaffold manufacturing techniques

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Scaffold manufacturing techniques
• This technique is known as gas foaming technique because it uses a foaming
agent (like sodium bicarbonate) into the polymer
• High-pressure gas is applied for disks of polymer in order to prompt the
nucleation and development of gas rise within the subjected substance
• They are afterwards lyophilized to obtain scaffold characterized with pore
diameters of about 100 μm and percentage porosity up to 93% upon the gas
liberation (like as N2 or CO2)

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Scaffold manufacturing techniques

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

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Scaffold manufacturing techniques

Thermally induced phase separation

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Scaffold manufacturing techniques

Thermally induced phase separation

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Scaffold manufacturing techniques
• This procedure depends on a thermal energy difference that initiates a
detachment of a polymer solution with a homogeneous network into a multi-
stage system domain using a quench method
• In the beginning, a high temperature is utilized to a solubilized polymer (either
in phenol or naphthalene), trailed by scattering of naturally dynamic particle in
these solutions
• Upon the temperature decrease the segregation is prompted, the resulted
solution isolates into polymer-free phase and solvent-free phase either by
strong fluid de-blending or fluid stage division component
• This step is then followed by extraction, dissipation, and sublimation in order to
eliminate the used solvent, thus, resulting in 3D polymer matrix impregnated
with bioactive particles coordinated within this porous structure.
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Scaffold manufacturing techniques

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Scaffold manufacturing techniques
• 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
scaffold with numerous and interconnected pores.

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Scaffold manufacturing techniques
• This technique has been utilized to fabricate a BG-Collagen-Phosphatidylserine
scaffold with interconnected pores with sizes of about 300 μm
• Phosphatidylserine, like several phospholipids, has been demonstrated to be
able to form complexes with both calcium and phosphate and nucleate HA
formation
• The scaffold was seeded with rat MSCs and either cultured for few days or
implanted in rat femurs defects
• In vitro results at day 21 showed that cells had grown within the scaffold,
showing osteogenic features
• In vivo results showed good biocompatibility and extensive osteoconductivity
with host bone after 6 weeks

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Scaffold manufacturing techniques
Disadvantages of this technique are the
lengthy timescales, high energy consumption,
the use of cytotoxic solvents and the
production of irregular, small size pores
(typically ranging from 15 to 35 μm)

SEM micrographs of the surface morphology of BG-COL-

PS scaffolds (A) the cross-section of the scaffold with a
structure of irregular interconnected pores ranged from
several microns up to about 300 μm (B) the
microstructure of the scaffold (C) the micropores located
on the pore walls of macropores in the scaffold.

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Scaffold manufacturing techniques
• 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

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Scaffold manufacturing techniques

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Scaffold manufacturing techniques

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Scaffold manufacturing techniques
• Rapid prototyping and additive manufacturing, also known as 3Dprinting, is a
strategy that was first portrayed in the year 1986
• The classical name of rapid prototyping strategy is stereolithography (SL) in
which thin layers of materials are deposited over each other by hardening the
layers utilizing ultraviolet waves

• Similarity, another strategy was created in particular Selective Laser Sintering

(SLS) to make 3D-structures
• SLS utilizes a powerful laser to sinter polymer powders to create 3D-structures

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Scaffold manufacturing techniques
• Sun et al. developed a novel MgO-CaO-SiO2 system by adding wollastonite
(CaSiO3) into Mg2SiO4 to fabricate bone scaffolds by SLS
• The scaffolds exhibited increased mechanical properties, a well-interconnected
porous structure, and the ability to support cell adhesion and proliferation

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Scaffold properties

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Scaffold properties
Comparison between different FFF parameters, infill (30%, 50% and 70%)
Comparison between different scaffolds geometries, layer thickness and porosities
and layer thickness (200 ​μm and 300 ​μm) regarding compressive modulus, a)
regarding compressive modulus, a) Ortho; b) 2xOrtho; c) Displ and d) 2xDispl
45 ​mm/s printing speed and 220 °C printing temperature; b) 30 ​mm/s and
scaffolds.
220 °C; c) 45 ​mm/s and 200 °C and d) 30 ​mm/s and 200 °C ortho scaffolds.

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Scaffold properties
Illustrative FE A example demonstrates the effect of
aligned (top row of images) and staggered (bottom
row of image) filaments on mechanical stiffness.
The four views from left to right indicate the 3D
model followed by side views (from left to right):
before compression, after compression, and a von
Mises stress colour map. The same force is applied
to both scaffolds in FE A simulations. In the top
scaffold, the filaments are aligned from top-to-
bottom and therefore form a continuous pillar of
polymer that resists compression. In contrast, the
bottom scaffold has staggered filaments and the
structure compresses by deformation at hinge
points (located at regions of high-stress
concentration in the von Mises stress plot). The
scaffold collapses in a concertina manner by slightly
bending filaments, which results in reduced stiffness
versus the top scaffold with a continuous column of
polymer.
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Scaffold properties

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Scaffold properties

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Scaffold properties

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Conclusions
• Although the rapid evolution in BTE, there
are still challenges to face that hamper the
progress towards clinical application and
new approaches need to be actively pursued
• These challenges will drive future
developments in the field
• The starting point is the identification of
the proper scaffold-based BTE therapy,
including the choice of material’s properties
and manufacturing methods as well as of a
multi- vs. single-component treatment
• Around this nucleus revolve a series of
important step elements: pre-clinical in vitro
and in vivo investigations, clinical trial
approval and conduction, scaffold
commercialization and patient expectations

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