Page 208 - IJB-10-5

International Journal of Bioprinting Biomimetic osteochondral scaffold

aggregates can mimic the microstructure of cartilage 4 μg BMP-2 per g scaffold, respectively) was subsequently
lacunae and are probably more suitable for cartilage added into the emulsion and stirred for 20 min, resulting in
regeneration than discrete cells. the water-in-oil bioink for the subchondral layer. Secondly,
the bioink for the interface layer was prepared the same as
In this study, we developed a tri-phasic scaffold with
a biomimetic structure and a spatiotemporally controlled the subchondral layer except without BMP-2. Thirdly, 6 g
P(DLLA-TMC) was fully dissolved into 9 mL DCM under
delivery capability of BMP-2 and FGF-18 via cryogenic
3D bioprinting, followed by the infusion of gelatin 20 min of ultra-sonication. Approximately 1 mL DI water
methacrylate (GelMA) hydrogel in the cartilage layer. A containing FGF-18 (final concentration of 1, 2, 3, and 4 μg
poly(lactic-co-glycolic acid)/tricalcium phosphate (PLGA/ FGF-18 per g scaffold, respectively) was then added into
TCP) interface layer was printed between the subchondral the P(DLLA-TMC)/DCM emulsion and stirred for 20 min,
layer and cartilage layer to prevent the mutual diffusion of resulting in the bioink for the cartilage layer. Finally, 5%
BMP-2 and FGF-18. Thereafter, discrete MSCs were seeded GelMA hydrogel precursor with 0.2% LAP photoinitiator
in the subchondral layer while MSC microspheres were was added to fill the macropores of the cartilage layer
seeded in the cartilage layer. The spatiotemporal delivery with hydrogels.
of BMP-2 and FGF-18 in the respective layers facilitated 2.3. Cryogenic 3D bioprinting of
the osteogenic differentiation of discrete MSCs in the osteochondral scaffolds
subchondral layer and the chondrogenic differentiation of A digital stereolithography (STL) file was imported
MSC microspheres in the cartilage layer, respectively. This into a cryogenic 3D bioprinter, and scaffolds with a grid
study provided an innovative strategy for constructing pattern and gradient structure were subsequently printed.
engineered biomimetic cell-laden osteochondral tissue Typically, four layers of subchondral struts were printed
with desirable MSC differentiation endpoints and displayed first, followed by two layers of interface struts and four
great potential for osteochondral regeneration. layers of cartilage struts. The subchondral and cartilage
layers contained parallel rods with a length of 10 mm and a
2. Materials and methods diameter of 0.4 mm; the distance between the two parallel
2.1. Materials rods was 0.8 mm. In contrast, the interface layer contained
Poly(lactic-co-glycolic acid) (PLGA) (lactic acid parallel rods with a length of 10 mm and a diameter of 0.2
[LA]:glycolic acid [GA] = 50:50) and shape-memory poly mm; the distance between the two parallel rods was 0.2
(d,l-lactic acid-co-trimethylene carbonate) (P[DLLA- mm. All the above rods in adjacent layers had a cross angle
TMC]) (DLLA:TMC = 80:20) were obtained from Jinan of 90°. Furthermore, the above biofabricated scaffolds
Daigang Biotechnology Ltd. (China). β-tricalcium were lyophilized for 24 h to remove DCM. After the
phosphate particles (β-TCP) were purchased from Aladdin production of the osteochondral frame, GelMA hydrogel
(China). BMP-2 and FGF-18 were Beyotime products was dispensed into the macropores of the cartilage frame,
(China). Dulbecco’s Modified Eagle Medium (DMEM), followed by photopolymerizing at 365 nm ultraviolet (UV)
2
Dulbecco’s Phosphate Buffered Saline (DPBS), fetal light with light intensity 12 mW/cm for 15 s.
bovine serum (FBS), penicillin (100 U/mL), streptomycin
(100 U/mL), and bovine serum albumin (BSA) were 2.4. Characterization of osteochondral scaffolds
purchased from Gibco (United States of America [USA]). Scanning electron microscopy (SEM, S-3000N, Hitachi,
Dichloromethane (DCM) was obtained from Macklin Japan) was employed to observe the microscopic
(China). Gelatin (derived from porcine skin; analytical morphology of these scaffolds at a voltage of 5 kV after
grade; 99% pure), methacrylic anhydride (MA) (94%), lyophilization and gold plating (thickness: 10 nm).
and lithium phenyl-2, 4, 6-trimethyl benzoyl phosphinate Compression testing was used to measure the mechanical
(LAP) were purchased from Sigma-Aldrich (USA). properties of osteochondral scaffolds under wet conditions
at 37°C. Specifically, three samples for each type of scaffold
2.2. Preparation of bioinks for (5 × 5 × 5 mm ) were tested, and the strain speed of 1 mm/
3
osteochondral scaffolds min was adopted. The in vitro degradation of scaffolds was
Three different bioinks were prepared for the biofabrication investigated by measuring the remaining weight (%) within
of biomimetic osteochondral scaffolds. Firstly, 3 g PLGA an 8-week test period. Typically, 100 mg of each sample was
was dissolved completely into 9 mL DCM, and 3 g TCP immersed in simulated body fluid (SBF) in tubes under a
particles were then added into the solution above to obtain shaking water bath at 37°C and 80 rpm. At each time point
uniform a β-TCP/PLGA/DCM emulsion after 20 min of (2, 4, 6, and 8 weeks), the test samples were taken out and
ultra-sonication. Approximately 1 mL of deionized (DI) lyophilized for 48 h, after which the remaining weight was
water containing BMP-2 (final concentration of 1, 2, 3, and calculated.

Volume 10 Issue 5 (2024) 200 doi: 10.36922/ijb.3229