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International Journal of Bioprinting CS-laden microporous bio-ink for cartilage regeneration

glycosaminoglycan (GAG) total content dimethylmethylene Finally, agarose microwell molds with 2750 microwells were
blue (DMMB) kit was purchased from GENMED transferred into the wells of the 6-well plate, and a little 2%
Scientifics (Boston, USA). Hydroxyproline (HYP) assay agarose was added around the mold for immobilization.
kit was purchased from Nanjing Jiancheng Bioengineering These microwell molds were rinsed with DPBS and stored
(Nanjing, China). Histostain-Plus Immunohistochemical in a 37°C incubator until use.
Kit was purchased from NeoBioscience (Shenzhen, China). For the formation of CSs, chondrocytes were harvested
LightCycler 480 SYBR Green I Master was purchased from and resuspended, then 1 mL of the appropriate density of
Roche (Basel, Switzerland). Oligo(dT)15 Primer, M-MLV cell suspension was added dropwise into each microwell
Reverse Transcriptase, and dNTP Mix were purchased mold. To prepare CSs with different cell numbers, three
from Promega Corporation (Wisconsin, USA). cell densities were used: 0.55 × 10 , 1.375 × 10 , and 2.75 ×
6
6
New Zealand white rabbits (male, 4 months old) were 10 cells/mL, resulting in approximately 200, 500, and 1000
6
purchased from Beijing Long’an Experimental Animal cells per microwell. After the cells had settled into the
Breeding Center (Beijing, China). BALB/c nude mice microwells due to gravity, 4 mL of complete medium was
(female, 6 weeks old) were purchased from Beijing Vital carefully supplemented into each microwell mold, and these
River Laboratory Animal Technology Co., Ltd (Beijing, 6-well plates were incubated at 37°C with 95% humidity
China). Animal experiments were approved by the Animal and 5% CO . Half of the medium was refreshed every 2
2
Care and Experiment Committee of Plastic Surgery days. Images of each group (200, 500, and 1000 cells/CSs)
Hospital. were taken with a light microscope (T12-U, Nikon, Tokyo,
Japan) at 0, 1, 7, and 14 days of culture for morphological
2.2. Isolation and expansion of auricular evaluation. These images were processed with ImageJ
chondrocytes software (version 1.53c, NI Health, Bethesda, MD, USA),
Chondrocytes were obtained from the auricle of the New and the diameters and areas of the CSs were measured (n =
Zealand white rabbits. The auricular cartilage was minced 100). The process of non-adhesive microwells preparation
into 1–2 mm pieces, after removing the soft tissue and and CSs formation is illustrated in Figure 1.
3
perichondrium. Following this, the pieces were digested
with 0.25% trypsin-EDTA for 30 min and 0.2% type II 2.4. Viability, histology, and biochemical analysis
collagenase for 6–8 h under continuous agitation at of CSs
37°C. The obtained cells were cultured and expanded in After 1, 7, and 14 days of culture in microwells, the CSs
a complete medium (high-glucose DMEM supplemented were collected for viability, histology, and biochemical
with 10% FBS and 1% PSN) at 37°C with 95% humidity analysis. The viability of the CSs was assessed using live/
and 5% CO . Chondrocytes in passage 2 were used for dead staining. Briefly, CSs were rinsed with DPBS and
2
further experiments. incubated for 20 min with Calcein-AM (2 μM) and PI
(4.5 μM). In addition, to facilitate the observation of cells
2.3. Preparation of non-adhesive microwells, within CSs, DAPI was used to label nuclei according to
formation, and culture of CSs the manufacturer’s instructions. These CSs were imaged
Non-adherent microwells were fabricated by a double- with an inverted fluorescence microscope (T12-U, Nikon,
molding procedure. At first, disc-shaped master mold with Tokyo, Japan).
a diameter of 30 mm and a height of 3 mm, consisting
of 2750 microwells with 400-μm diameter for each well, For histology analysis, CSs were fixed in 4%
was designed using AutoCAD (Autodesk, 2022). Each paraformaldehyde for 1 day, pre-embedded with 0.5%
25
microwell comprised a 200-μm deep cylindrical section agarose (w/v)-1% gelatin (w/v) mixture, dehydrated
and a 200-μm deep hemispherical bottom. According to in graded alcohol, embedded in paraffin, and sectioned.
the design, polytetrafluoroethylene (PTFE) was processed Subsequently, sections were stained with hematoxylin and
by computer numerical control (CNC) machine tools eosin (H&E) and Alcian blue to evaluate the histological
(T-600S, Taikan, Shenzhen, China) to obtain microporous structure and glycosaminoglycan (GAG) deposition in the
master molds. Next, the PDMS was poured into the PTFE CSs. All histological images were obtained using a digital
molds, cured at 65°C for 2 h after degassing, and demolded slice scanner (EasyScan 1, Motic, Xiamen, China).
from them. Then, the sterilized PDMS micropillar molds At each time point, CSs in three independent agarose
were placed in a petri dish, and 2% (w/v) ultrapure agarose molds per group (n = 3, each containing 2750 CSs) were
dissolved in DPBS was heated and poured onto the PDMS collected separately and subjected to DNA and GAG
molds. Once the agarose molds had solidified after cooling content measurements. The contents of DNA and GAG
at room temperature, they were punched into a size (33-mm were measured using Quant-iT™ PicoGreen® dsDNA assay
diameter) to fit 6-well plates and separated from the PDMS. and DMMB assay, respectively.

Volume 10 Issue 1 (2024) 201 https://doi.org/10.36922/ijb.0161