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Morphological, mechanical and biological assessment of PCL/pristine graphene scaffolds for bone regeneration

life. It is estimated that in Europe 179,000 men and Its capacity to stimulate cells is also another impor-
611,000 women will suffer hip fracture each year and tant requirement. As electrical signals are critical phy-
the cost of all osteoporotic fractures in the EU will siological stimuli that strongly affect cell behavior,
increase from the current 31.7 billion euros to 76.7 electro-active scaffolds could have a great potential as
[4]
billion euros by 2050 . substrates for tissue engineering, enabling cell stimu-
In these cases, the clinical approach is the use of lation, as well as increasing their proliferation and
bone grafts, defined as an implanted material that pro- differentiation [17–20] . In order to produce these scaf-
motes bone healing alone or in combination with other folds, different routes are explored, including the use
materials, through osteogenesis, osteoinduction, and of conductive polymers mixed with non-conductive
[5]
osteoconduction . Bone grafts can be divided into polymers, and the use of inorganic conductive materi-
autografts, allografts, and xenografts [1,2,6] . However, als with non-conductive polymers. Pristine graphene
there are many inherent limitations with this proce- (highly pure graphene material) is a two-dimensional
dure. Autografts are considered to be the most effec- carbon nano-filler that can be used to create elec-
tive approach, however, they present some drawbacks tro-active scaffolds, with potential to improve me-
such as site morbidity, pain, and prolonged hospitali- chanical and conductivity properties. Different manu-
sation [2,3] . Allografts are associated with rejection pro- facturing techniques, like solvent precipitation/casting
blems, transmission of diseases and infections from and electrospinning, have been used to produce gra-
donor to recipient, and cost [1,2,6] . Xenografts major lim- phene composites substrates (2D), as well as foams
itations are related to their lack of osteogenic proper- and scaffolds (3D) [21–23] . However, these techniques
ties, risk of immunogenicity and transmission of in- are not fully reproducible and do not allow a good
fections and zoonotic diseases, and poor clinical out- control over pore shape, size, and interconnectivity,
come [5,6] . Therefore, biofabrication—the combined use which are critical parameters to design optimised 3D
of additive manufacturing techniques, biocompatible scaffolds. Additionally, a number of studies reported
and biodegradable materials, cells, growth factors, etc., on the cytotoxicity of graphene-based composite ma-
for the fabrication of bioactive scaffolds (synthetic terials and its potential risks [24–26] , while others re-
grafts)—is becoming a promising alternative for graf- ported that graphene-coated surfaces presented good
ting [7–14] . In this approach, scaffolds provide an initial cytocompatibility, stimulating cell proliferation [27,28] .
biochemical substrate for the novel tissue until cells This paper investigates the potential usage of PCL/
can produce their own extra-cellular matrix. An ideal pristine graphene scaffolds, containing very small con-
scaffold for bone tissue engineering must be designed centrations of pristine graphene (to avoid potential
according to the following requirements [3,10,11,15,16] : cytotoxicity effects), for tissue engineering applica-
 The scaffold material must be non-toxic and allow tions. Two major effects were considered; how effec-
cell attachment, proliferation, and differentiation; tive is pristine graphene to improve the mechanical
 The scaffold material must degrade into non-toxic properties even in small concentrations, and the effect
products under a controlled degradation rate; of small concentrations of pristine graphene on both
 The scaffolds should promote osteointegration, cell viability and proliferation. Scaffolds with different
which corresponds to the formation of a chemical material compositions were produced using an extru-
bond between bone and the surface of the implanted
scaffold without the formation of fibrous tissue. sion-based additive manufacturing technique, which
They must also promote osteoconduction and os- allows high reproducibility and the fabrication of
teogenesis, inducing chemical stimulation of human scaffolds with good control over its topology (pore
mesenchymal stem cells into bone-forming os- size, shape, distribution, etc.).
teoblasts;
 Scaffolds must be able to deliver growth factors, 2. Materials and Methods
cytokines, and antibacterial materials. 2.1 Materials
Scaffolds must present sufficient strength and stiff-
ness to withstand stresses in the host tissue environ- Poly (ε-caprolactone)
ment and adequate surface properties like wettability Poly (ε-caprolactone) (PCL) used in the research was
and surface roughness guaranteeing that a good bio- Capa 6500 (Perstorp, UK). PCL is a semi-crystalline
mechanical coupling is achieved between the scaffold biocompatible and biodegradable linear aliphatic
and the tissue. polyester with a low melting point and glass transition

96 International Journal of Bioprinting (2016)–Volume 2, Issue 2