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University of Minho

Advanced Biomaterials and Biomimetics

4th Year 2nd Semester

Hierarchical Structures Mimicking


Braga, 9th May, 2017

Introduction ......................................................................................................................................................... 3
Bone..................................................................................................................................................................... 4
Tissue Engineering for Bone Regeneration ......................................................................................................... 6
Processing hierarchical materials ........................................................................................................................ 7
Literature Review and Proposed Application ...................................................................................................... 9
References ......................................................................................................................................................... 12
Natural structural materials are developed at ambient temperature with specific components.
The bone is included in this category of materials were theres a distinction between hard and soft
phases, arranged in complex hierarchical1 structures. Mimicking the properties of such materials
and creating artificial ones has proven to be a tough challenge [1].
A lot of natural structures are recognized as lightweight offering high mechanical properties.
The designer should be aware that strength and toughness of the material are related. Usually,
strong materials tend to be brittle, while tough materials are sometimes weak. Thats why natural
materials offer great possibilities for insight and development of new structures. For instance,
organisms reveal porous architectures that combine light weight and stiffness such as cancellous
bone. To acquire strength, some materials feature architectural gradients and incorporation of
nanofibers [1].
To understand the relation between stiffness and strength for different families of materials,
is possible to use Ashby plots, as seen in Figure 1.

Figure 1 Ashby plot normalized by density including both natural and synthetic polymers [1]. Each closed region represents a family of

As observed, ceramics and metallic alloys have values of strength considerably higher than
regular natural materials. One would say that natural materials dont offer extraordinary properties,
nevertheless these materials are built from inexpensive constituents like proteins, polysaccharides,

In this context, hierarchical refers to the gradual presence of features from the nano scale, building up to the microscale and so
forth, reaching the final tissue.
calcites and rarely metals. Also, many of these materials have the ability to repair themselves after
damage, in contrast to synthetic materials who generally wont self-heal [1].

Bone is an example of a natural material that combines both strength and toughness, being
considered damage-tolerant. It is composed of cells embedded in a extracellular matrix (ECM), an
ordered network where it can be distinguished two nanophases: collagen fibrils made from type-I
collagen molecules (~300nm long, ~1.5nm in diameter) and hydroxyapatite (10 (4 )6 ()2)
nanocrystals (plate-shaped, 50nm 25nm, 1.5-5nm thick) dispersed along the collagen fibrils (
Figure 2) [1]. Each of these components are present at different scales ranging from nano to
micro scale with different orientations [2].

Figure 2 Hierarchical structure of bone [1].

Macroscopically, two types of bony tissue arrangements are differentiated: compact/cortical

bone at the surface, creating a shell, and spongy/trabecular bone (foam-like material, also
designated cancellous bone). Despite of having similar composition, the hierarchical structures lead
to different mechanical properties [2][1].
The trabecular bone has a highly porous structure (90% porosity), and at the mesoscopic
level it is composed of plates (trabeculae, 100-300 m thick with spacing of 300-1500 m between
adjacent trabeculae), positioned next to small cavities containing red bone marrow [2]. Collagen-
Hydroxyapatite composite materials, with properties similar to that of trabecular bone can be used
for tissue engineering applications [1].

Cortical bone has only 10% porosity and is constituted by osteons and Haversian canals
(central canals), which surround blood vessels. Osteons can reach 200 m in diameter and 10 to
20 mm long. They have a lamellar structure, where each individual lamella consists of organised
and fibres, creating patterns. The fibres include mineralised collagen fibrils, consisting of collagen
proteins (tropocollagen) composed of three chains of amino acids and nanocrystals of
hydroxyapatite (HA), linked by an organic compound to form fibril arrays. In this bony tissue cells
are not found beyond 200 m of a blood and oxygen supplies. Due to this fact, porosity and presence
of channels, that help cell migration and vascularisation, should be considered during scaffold
design [1].
Usually, hydroxyapatite crystals are oriented with the c axis parallel to the collagen fibrils,
being disposed regularly along the fibrils. This structure forms a light-weight, self-healing
multifunctional material.
The following table compares the properties of both cortical and trabecular bones, including
pore size and proportion.

Table 1 Comparison between cortical and trabecular bone [1].

Rigid, Dense (10% porosity), Highly porous (90% porosity),
consists of osteons, oriented in consists of small interconnected
longitudinal direction in long bones. bone trusses (trabeculae), oriented
along principal stresses directions.
Harversian canals with 0.028-0.061 ~1mm in diameter
Pore size mm in diameter

Proportion 80% of total mass 20%

(varies according
to location)
Anisotropic, depending on osteon Modulus of elasticity and ultimate
Mechanical orientation. compressive strength is about 10
properties Improves the mechanical properties times lower than cortical.
of trabecular bone.

Fracture resistance in the cortical bone is determined by two mechanisms: intrinsic
mechanisms that lead to ductility and extrinsic that prevents crack propagation.

Tissue Engineering for Bone Regeneration

Musculoskeletal tissue engineering (TE) including regenerative systems for cartilage,
ligament and bone can offer a solution for osteoporosis, osteoarthritis, trauma-induced injuries and
osteochondral lesions [2].
In particular, TE is associated with in vitro seeding and attachment of human cells onto a
scaffold, creating a microenvironment for proliferation, migration and differentiation of cells into a
particular tissue.
To provide the support for cellular proliferation and proper bone regeneration, scaffolds must
be designed to provide a hierarchical structure. Since cells respond to physiochemical properties of
the matrix by using their receptors, the molecular characteristics of the scaffold (e.g. collagen type)
and microscopic features (e.g. microstructure and porosity) can affect cell behaviour.
At the molecular level, characteristics such as collagen type, its cross cross-linking and HA
stoichiometry will determine if the biomimetic collagen-HA scaffold is suitable.
Collagen comprises many ligands and growth factors that promote cell attachment and
proliferation. As mentioned before type I tropocollagen self-assemble to form collagen fibrils, which
in turn form larger fibres. Much of the collagen materials are extracted from animal tissue derivatives,
leading to issues of antigenicity. Hence atelocollagens are the preferred form of collagen for
applications of biomimetic bone TE [2].
On the other hand, starch-based materials have been widely used as biomaterials for bone
replacement/ fixation and tissue engineering scaffolds. Starch is a polysaccharide that is
inexpensive, renewable and biodegradable. This material is composed of two polysaccharides:
amylose and amylopectin. The first one has linear structure and the latter is highly branched. It is
reported that this material is noncytotoxic and induces attachment and proliferation of osteoblast-
like cells [3].
Starch cannot be processed through conventional plastic technology, unless it is further
modified, since the degradation starts at a temperature lower than its melting point. Its It is possible
to decrease the glass transition temperature of starch by adding water or other plasticizers (e.g.
glycerol). Thermoplastic starch is highly hydrophilic, decreasing its performance and stability in
humid environments. According to literature, blending starch with other biodegradable and
biocompatible polymers is considered the most effective strategy to overcome this defect.
Polycaprolactone (PCL) is a hydrophobic, semi crystalline and biocompatible polymer that
can be prepared through ring opening polymerization of -caprolactone. This material has good
solubility, low melting point, and has extraordinary blend-compatibility. In contrast to starch, PCL
can easily be processed at relatively low temperatures and presents a total degradation of 2 to 4
years, depending on its molecular weight ( ). Random hydrolytic chain scission of the ester
linkages is the mechanism of PCL degradation that causes a decrease in its . This polymer is
suitable for drug delivery and tissue engineering, including bone tissue. Despite the good properties
the low melting point makes it difficult to process using conventional techniques, leading to high
production costs [3].
To overcome this issue, more recently, PCL and starch composites (SPCL) are being used
for tissue engineering. PCL can reduce the high sensitivity of starch to humid environments, improve
its processability and reduce its high stiffness. On the other side, starch is able to promote PCL
biodegradability and reduce the high cost of production. By changing the component ratio, one could
adjust the mechanical and degradation properties of SPCL.

Processing hierarchical materials

Despite of being difficult to mimic the complexity of biological materials, there are several
processing techniques that reach comparable results to whats presented in the nature. Methods
such as biomimetic mineralization and freeze casting can be used to create hierarchical structures.
In particular, for bone scaffolds, additive manufacturing (AM) is an emerging technique that allow to
build structures layer by layer using a computer aided design model (CAD). There is a wide range
of materials that can be used in AM for bone scaffold manufacturing, nevertheless PCL and PLA
(polylactic acid) are the most commonly used materials, due to the biodegradability, proper
mechanical properties and approval for medical implantation. Also, compared to natural polymers
both PCL and PLA yield higher strength and processability, but also controllable degradation rates
[4]. HA is another interesting material for bone scaffolds that can be included in AM by using
significantly higher processing temperatures than the ones used for PCL or PLA alone. Another
option is to build HA scaffolds by casting it to sacrificial moulds produced with AM. Both FDM (fused
deposition modelling) and SLS (selective laser sintering) are the most common AM techniques used
in bone tissue engineering applications.
Another emerging technique is 3D bioprinting which consists of extrusion of material from a
nozzle but unlike FDM it does not involve heating, thus it can utilize sensitive materials including
gels and cellular aggregates [5].
Usually the process starts with the oriented deposition of a gel filament, forming a grooved
layer. As with all 3D printing methods, a CAD model is initially created and then sent to the 3D

printer. The materials are deposited layer-by-layer, where each layer can encompass a combination
of different materials. In an inkjet printer, the bioplotter printer is capable of using and changing
bioink to develop the final scaffold structure (Figure 3a). 3D-Bioplotter systems (EnvisionTec,
GmbH) can print cell-laden gels, often combining other polymeric materials such as PCL, to produce
functional scaffolds. The printer utilizes a pneumatic pressurized system to extrude the material from
the bioink units [4]. The inconvenient of using this system is the developed shear stress from the
nozzles, which can negatively affect cell viability during the printing process. Despite of that, 3D
Bioplotting requires less time for scaffold preparation comparing to 3D Printing, since it avoids
a second posttreatment step [6].
Three main technologies used in 3D bioprinting are presented in Figure 3 [7].

Figure 3 (a) Thermal inkjet printers heat the printhead electrically to form air-pressure pulses conducting to droplets. Acoustic printers use
piezoeletric or ultrasound pulses. (b) Microextrusion printer uses pneumatic or mechanical systems to extrude continuous beads. (c) Laser-
assisted printer uses lasers focused on a substrate that propel cell-containing material, onto the collector substrate [7].

The following table summarizes state of the art bioprinters used for bone tissue engineering.
Its relevant to note that despite of being possible to bioprint bone tissue for critical-size defects,
scale-up vascularized bone tissues still remains intangible [8].

Table 2 Cell types, Bioprinters and printing parameters used in different studies for bone tissue 3D bioprinting [8].

Cell types Bioink/ Bioprinting Bioprinters

bioprinted substrate Technology Used
Bone-marrow- PEGDMA [9]; Thermal Inkjet [9]; Hewlett-
derived human nano-HA slurry laser-induced droplet Packard
mesenchymal [13]; Matrigel ejection [13]; deskjet [9]; HT-
stem cells [9]; and alginate [10]; extrusion-based BioLP
Endothelial BMP2 and fibrin (pneumatic) [10]; workstation
progenitor and (substrate) [11]; Piezoelectric Inkjet [13];
multipotent [11]; Bioplotter [12]
stromal cells [10];
[10]; MicroJet
Primary-muscle- [11];
derived stem
cells [11];
SaOs-2 cell line

Literature Review and Proposed Application

Sara Olveira et al. (2012) fabricated PCL hierarchical scaffolds using prototyping technique,
with a Bio-plotter, with strut diameters of approximately 0.5 mm and 10 consecutive layers, each
oriented at 90 degrees with respect to the underlying layer [12]. Nano/micro fibrillar structures of
alginate and chitosan were created inside 3D scaffolds, by controlling self-assembly parameters
(e.g. polyelectrolytes concentration and washing time) the distribution of this structures can be
controlled. The introduction of the fibrillar features improved cell seeding efficiency.
Regarding SPCL, Santos et al. produced scaffolds using fibre-bonding based in fibre obtained
by melt spinning. The resulting fibres were posteriorly positioned in a glass mould, heated in an
oven, compressed using a Teflon cylinder, and then cooled [3].
Martins et al. (2008) utilized a liquid-based 3D dispensing approach with molten polymer/
starch achieving a resolution of 300. The device used to fabricate the scaffold was the 3Dd-
Bioplotter, using a 30:70 (wt%) blend of starch and polycaprolactone (SPCL, Novamont, Italy).

Human osteosarcoma-derived cells (Saos-2 cell line) were maintained in Dulbeccos modified
Eagles medium, supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic-
antimycotic solution.
The hierarchical fibrous scaffold created (6RP + 5NFM), combined SPCL micro- and PCL
nano-motifs, respectively produced by rapid prototyping (RP) and electrospinning (ES) [14].
The 3D structure presented parallel aligned rapid prototyped microfibres (average fibre
diameter, 300 m), periodically intercalated by randomly distributed electrospun nanofibres (fibre
diameters in the range 400 nm1.4 m) (figure 1B). -CT analysis revealed that the integration of
the nano-motifs in the scaffold (6RP) decreased porosity from 79.4% to 68.3%.

Figure 4 A, C- SEM and -CT respectively, micrographs of the starch-based rapid prototyped scaffold, 6RP (without nano-motifs). C, D -
SEM and -CT respectively, micrographs of the hierarchical fibrous scaffold (6RP + 5NFM) [14].

The results shown that human osteoblast-like cells presented significantly higher proliferation
and maturation when seeded on the starch-based fibrous scaffolds, due to the biomimicry of the
scaffold with respect to the ECM [14].
Another similar study were two techniques were combined to create an improved scaffold,
was presented by Yu et al. (2014). In order to mimic the natural morphology of the ECM, 3D
bioprinting and electrospinning were used to produce gel microfibers and PCL nanofibers
respectively. In this study is reported that the main advantage of 3D bioprinting is that many
biomaterials can be used to produce complex structures with precise control of pore size. Similarly
to the last study, the results of cell culture revealed that the hierarchical PCL/gel scaffold can
improve initial cell attachment, cells growth and proliferation compared to the 3D bioprinting scaffold
Finally, Jukola et al. (2008) and more recently Komur et al. (2017) realized composite SPCL
nanofibers using electrospinning. In the latter, the samples were prepared using a co-axial needle
electrospinning technique, and the cell culture studies determined that the increase of starch in the
fibre also increases the cell viability, however further analysis may be required to demonstrate this
In conclusion, several methods reveal potential in bone tissue applications. As novel
technique, the combination of 3D bioprinting with SPCL nanofibers using electrospinning is
proposed to fabricate hierarchical scaffolds. Also, to promote osteoblasts differentiation and
migration, several growth factors can be included in the scaffolds, such as BMP-2 and BMP-7 [16].

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