(496g) Nanofibrous Scaffolds Produced By Electrospinning, Rotary-Jet Spinning and Airbrush for Orthopedic Tissue Regeneration

Despite
the progress tissue engineering has made in the development of improved biomaterials,
inhibiting bacterial infection has not been a central focus to date. Infection
is a leading cause of implant failures with many
agencies (such as the Centers for Disease Control) predicting more deaths from
bacteria than all cancers combined by 2050. Gram-negative
bacteria are naturally resistant to numerous treatments and are difficult to kill
due to their robust and hydrophobic outer lipopolysaccharide membrane which
helps to prevent the flow of antibiotics or drugs into the cell. Moreover, due
to extensive antibiotic use, gram-positive Staphylococcus aureus has
evolved to a methicillin-resistant strain, which can overcome other classes of
antibiotic treatments. The development
of an implant capable of reducing bacterial growth (without resorting to the
use of antibiotics, which causes antibiotic resistant bacteria) would be an
effective way to improve implant success. Recently, scientists have been
investigating novel materials and techniques to meet growing
orthopedic tissue engineering needs. Due to problems (such as infection and
long healing time) that titanium based implants cause in vivo, new
polymeric materials have been introduced. The first step in implant infection is bacterial adhesion, which can
potentially result in the formation of antibiotic resistant biofilms for some
species. Bacterial adhesion, growth, and subsequent biofilm
formation on surfaces are particularly resistant towards the body’s defense
mechanisms and antibiotic treatments, which can cause implant rejection. Multiple substrate properties, including
chemical composition, hydrophobicity, and surface roughness, are believed to be
of significance in the bacterial attachment process. Recently,
researchers have been exploring alternative physical methods through reduced
contact of microorganisms with an implant which would prevent extensive
bacteria proliferation on the surface and allow the immune system to clear such
bacteria. Materials with
biologically-inspired nanostructured features have shown promise to reduce
bacteria growth. Polycaprolactone
(PCL) is a bioresorbable and biocompatible polymer with potential applications
for bone and cartilage repair. In this study,
polycaprolactone fibers (with and without hydroxyapatite nanoparticles (nHAp)
and carbon nanotubes (CNT)) were produced using three different methods:
electrospinning, rotary-jet spinning and airbrush. The scaffolds were characterized
using contact angles, differential scanning calorimetry, scanning electron
microscopy, and were subjected to cell culture, bacterial assays, and
mechanical (tensile) testing. Early interactions between the surface of the
material with biological fluids play a fundamental role in determining cellular
behavior (such as adhesion, differentiation, and tissue formation). The
biological and material properties were studied to understand how the various
fabrication techniques and nanoparticles affect human osteoblasts, gram-positive
(Staphylococcus Aureus) and gram-negative (Pseudomonas aeruginosa)
bacteria growth on samples. Experiments showed no toxic effect on osteoblast
cells and a significant decrease in bacterial density by adding nHAp and CNT to
the PCL scaffolds without using growth factors or antibiotics. Most
importantly, results showed that without the use of antibiotics, by only changing the
fabrication method and the size of the fibers, antimicrobial properties were
improved without sacrificing mechanical properties and osteoblast
functions. Among the different fabrication methods, airbrushed fibers indicated
the highest elastic modulus compared to the other two techniques. Specifically,
it was found that the antimicrobial properties were improved when using
airbrushing compared to electrospun and rotary-jet
spun scaffolds, where up to a 50% decrease in bacterial density in both gram-positive
and gram-negative species was observed (Fig. 1). To assess osteoblast
biocompatibility and differentiation, cell proliferation, viability, adhesion,
and calcium deposition assays were conducted. Although the cell proliferation,
calcium deposition and cell viability results are similar, it is observed
that the cell adhesion in airbrushed samples are poorer than those in
electrospun and rotary-jet spun scaffolds. To investigate the influence of
fibers’ morphology on osteoblast cells, further gene expression assay (qPCR)
will be conducted; which means based on the cell culture assays that have
already been done, we should be looking for specific gene expressions such as alkaline
phosphatase, and osteocalcin and compare them to the results from mammalian
cell culture from different fibers. In other words, the future dream of this
study is to demonstrate how different fabrication methods and hence various
surface properties can make cells behave differently. It is of significance to
mention that PCL fibers cannot compete with metallic implants such as titanium and
stainless steel strength-wise. The application of this study is to heal the
defects in bone/cartilage by increasing the tissue growth and minimizing bacterial
colonization without use of any growth factor or antibiotic.

Figure
1. All samples
contain 1% of CNT and 1% of nHAp. Colony Forming Unit (CFU) method was used to
measure the density of bacteria.  a) Staphylococcus
aureus density, and b) Pseudomonas
aeruginosa density in different samples. Values are mean ± SD, N=3, * p < 0.05, ** p < 0.1, *** p < 0.01, **** p <
0.001 compared to each other.