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International Journal of Bioprinting 3D-printed variable stiffness scaffolds

phenylindole dihydrochloride) was used to stain cell nuclei. endo-peroxidase activity. Sections were washed, followed
Samples were fixed by immersing in 4% paraformaldehyde by incubation with a secondary antibody (Anti-IgG mouse;
(PFA) and incubating overnight at 4°C. Prior to staining, B7151; Sigma-Aldrich, USA), diluted to 1.5:200 using
the scaffolds were cross-sectioned and permeabilized in blocking buffer, for 1 h at room temperature. Sections were
0.5% Triton-X for 20 min at room temperature. For actin washed again and incubated with ABC reagent (Elite kit
staining, scaffold cross-sectional slices were incubated with Vectastain PK-6100; Vector Labs, USA). After washing with
the fluorescent agent rhodamine-conjugated phalloidin PBS, DAB (3,3-diaminobenzidine; Vector Labs, USA) was
(dilution 1:40; VWR, USA) for 1 h, followed by incubation used to detect the specific antibody reaction, indicated by
with DAPI (dilution 1:50; VWR) for 10 min under brown staining. Each section was washed with cold water
light protection. Samples were imaged using a confocal to stop the reaction, and sections were dehydrated through
microscope (FV-1000 Point Scanning Microscope; an alcohol series and soaked in xylene. Stained sections
Olympus, Japan) at the following absorption/emission were imaged using a microscope (BX60; Olympus, Japan).
wavelengths: rhodamine-phalloidin: 540 nm/565 nm;
DAPI: 358 nm/461 nm. 2.12. Biochemical assays
For biochemical analysis, constructs were digested with
2.11. Histology and immunohistochemistry papain (0.1 mg/mL; pH 6.4) in a sodium phosphate
Histology and immunohistochemistry analysis were buffer, containing 0.1 M sodium acetate, 5 nM L-cysteine
performed for day 1 (D1) and day 21 (D21) constructs HCl, and 0.05 M ethylenediaminetetraacetic acid (all
to confirm fibro-chondrogenic differentiation of the Sigma-Aldrich), overnight at 60°C with shaking at 100
seeded cells. D1 samples display the background staining rpm. However, it was found that samples containing
of the hydrogel and serve as a reference. Constructs were HAMA were not digested using papain alone. Therefore,
washed with PBS and then fixed by immersing in 4% PFA a hyaluronidase solution was subsequently used for the
and incubating overnight at 4°C. Samples were removed digestion of constructs containing HAMA, and an adapted
from PFA, washed, and stored in PBS at 4°C. At the time protocol by Beck et al. was used. Briefly, a 1000 U/mL
30
of the analysis, samples were dehydrated using a series hyaluronidase (Sigma-Aldrich, USA) solution was prepared
of ethanol (30%, 50%, 70%, 80%, 90%, 100%, xylene) using 0.02 M phosphate buffer containing 77 mM sodium
and wax embedded thereafter. Paraffin wax-embedded chloride and 0.01% BSA. Samples were allowed to digest
constructs were sectioned using a microtome (Leica, overnight at 37°C. Fresh papain solution was then added,
Germany) to produce 6-µm-thick slices and mounted on and constructs were allowed to digest overnight at 60°C.
microscope slides. Samples were then deparaffinized and Both the first and second digestion solutions were stored
rehydrated using a guided series of xylene and alcohol at −20°C until further analysis. Digests were analyzed on
baths. New tissue formation was determined by staining days 1 (D1) and 21 (D21), and media samples were taken
with hematoxylin and eosin (H&E). Sulfated GAG (sGAG) and analyzed across cell culture time points (days 1 [D1],
deposition was stained using Alcian blue, and secreted 7 [D7], 10 [D10], 14 [D14], 17 [D17], and 21 [D21]) for
collagen was stained using Picro Sirius Red (all from GAG and collagen content. The sGAG content in each
Sigma-Aldrich, USA). Following staining, the samples sample was quantified using a dimethylmethylene blue
were imaged using an Olympus CKX53 microscope dye-binding assay (Blyscan™ Glycosaminoglycan Assay;
(Olympus, Japan). Immunohistochemistry was conducted Biocolor, UK), as per the manufacturer’s instructions,
using the antigen retrieval method on constructs after 21 followed by measuring the absorbance using a microplate
days of in vitro culture to determine the type of collagen reader (Synergy Mx; BioTek, USA) set at 656 nm. To
synthesized. Sections were deparaffinized and rehydrated obtain the total biochemical content for each sample, the
using a series of xylene and alcohol baths. Briefly, sections two digestions (papain and hyaluronidase) and media were
were treated with peroxidase, followed by treatment with quantified and added together. Media samples were taken
chondroitinase ABC (0.25 U/mL) (Sigma-Aldrich, USA) at days of media change and were analyzed with the same
for 1 h at 37°C. Sections were incubated with goat serum to assays to track the release of biochemicals to the media.
block non-specific sites. Primary antibodies, anti-collagen
type I (mouse monoclonal IgG; 90395; Abcam, UK), were 2.13. Mechanical testing
diluted to 1:400 in blocking buffer; anti-collagen type II To determine the effect of cell growth on the mechanical
(mouse monoclonal IgG; 3092; Abcam, UK) was diluted properties of the ECM-like biomaterial within the
to 1:100 in blocking buffer. The sections were incubated construct, mechanical testing was performed using micro-
with the antibody overnight at 4°C. The sections were indentation between the PCL fibers. Stress relaxation
then washed in PBS and immersed in diluted hydrogen studies were performed using a standard testing machine
peroxide solution (3%; Sigma-Aldrich, USA) to block equipped with a 5 N load cell. For day 1 and day 21 samples,

Volume 10 Issue 4 (2024) 497 doi: 10.36922/ijb.3784