Page 170 - IJB-9-2

International Journal of Bioprinting 3D Printing Multifunctional Orthopedic Biocoatings

surface and reduce the incidence of charging, which is the viability of various human and animal cell lines . Cell
[64]
due to high negative charges accumulating on the sample viability and adhesion on these coated substrates were also
surface. SEM was also used to examine the nanocomposite assessed using live/dead staining (Invitrogen, Live/Dead
structure inside the dried polymeric coating surface. Staining Kit). The live and dead cells were visualized at
days 1 and 3 post-seeding using a fluorescence microscope
FTIR spectroscopy was performed on the sample
powders as well as on the obtained coating films using (Olympus-CKX41).
a Nicolet 6700 spectrophotometer (Thermo Electron 2.8. VA release measurements
Corporation) equipped with a diamond ATR Smart orbit
window. 2.8.1. Elution experiment
The coated substrates were placed in a sterile 24-well tissue
Spectra were obtained at 1.0 cm resolution averaging
−1
32 scans to investigate and confirm the presence of ACP culture plate with 2.0 ml of phosphate-buffered saline
(PBS, Lonza, 1×) completely covering the coating (n = 3
and VA within the polymeric coatings.
of each group) kept under 37°C, 5% CO , and 95% relative
2
2.6. Adhesion test humidity. All the 2.0 ml solution from the wells were taken
out and analyzed at each time point, and the wells were
The bond strength and stability of any coating on the replenished with 2.0 ml of fresh PBS. The concentration
substrate are a critical factor in determining its value to of VA released at each time point was determined by
biomedical applications. Low-quality films could peel- measuring the absorbance at 280 nm, from which the
off from the substrate when subjected to forces and concentration was calculated using a standard calibration
loads, and thus, it is important to evaluate their adhesion curve [64-67] . The mass of VA released was then calculated
properties. The adhesion of the polymeric coatings to the using the values of measured concentration and actual
Ti substrate was evaluated according to the American collected sample volume.
Society for Testing Materials (ASTM) (Mittal, 1978) .
[62]
ASTM-D3359-02 tape test was chosen to study the 2.8.2. Antibiotic activity study
[63]
adhesion of polymeric coatings on the Ti alloy substrates. The biological activity of the released VA was evaluated on
A crosscut pattern of 1 mm separation distance was made Staphylococcus aureus (S. aureus) by measuring the zone of
on the coating samples. An ASTM standard pressure inhibition using the disk diffusion method. The objective
sensitive tape was firmly adhered onto the coatings and of this test was to validate that the released VA is still
then removed according to the procedure as described in active after the coating on the Ti substrates. S. aureus was
the ASTM tape adhesion test. purchased from the American Type Culture Collection
2.7. Cell adhesion and cytocompatibility test (ATCC). S. aureus was pre-cultured with soy broth (BD
Biosciences, NJ) at 37°C in a shaking incubator for 7 h
To test the cytocompatibility of the various ACP polymeric and inoculated on a Mueller-Hinton agar plate. The blank
coatings, cell adhesion and live/dead tests were conducted. antimicrobial susceptibility disks (Oxoid, UK) were placed
The influence of factors, such as ACP concentration and on the bacteria plate and 10 µL of the elutes collected from
polymer type on osteoblast confluence and proliferation, each group at various time intervals were then carefully
was investigated. Murine osteoblast cell line, MC3T3-E1, loaded to these disks. The known concentrations of VA
was obtained from ATCC (Manassas, VA). Cells were were used for the control. The plates were incubated
cultured under 37°C, 5% CO , and 95% relative humidity overnight at 37°C and the area of microbial resistance was
2
in minimum essential medium (α-MEM, Gibco, Grand measured.
Island, NY) containing 10 vol.% fetal bovine serum (FBS,
Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin- 3. Results and discussion
streptomycin solution (P/S, Gibco, Grand Island, NY). 3.1. Coating solution and parameters
Cells at the third to seventh passage were used in this
experiment. All the substrates were sterilized under UV The custom developed 3D printing technique was employed
radiation for at least 60 min. The sterilized substrates were to coat all the substrates with different therapeutic agents in
placed in 12-well plates and MC3T3-E1 cells were seeded polymeric formulations. Monodispersed droplets as shown
on them at a concentration of 120,000 cells/well. A 1 ml of in Figure 2B were generated for polymeric solutions so that
media/cm of surface area was used and the culture media precision deposition was achieved on the Ti substrate.
2
were changed daily. The effect of ACP concentrations on The jetting performance of each candidate polymeric
the osteoblast viability was evaluated using the Alamar blue solution was dictated by the physical properties of the
assay. This bioassay was designed to quantitatively measure printing solutions, which were controlled by adjusting the

Volume 9 Issue 2 (2023) 162 https://doi.org/10.18063/ijb.v9i2.661