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International Journal of Bioprinting DEX-Loaded PLGA microspheres enhance cartilage regeneration

China). All animal experiments were conducted following We conducted a statistical analysis of the pore
approval from the Animal Care and Use Committee diameter of porous microspheres. Three representative
of the Plastic Surgery Hospital (Approval No: EAEC samples were randomly selected from each group, and
2022-007, EAEC-008). 30 pores were measured on each microsphere; therefore,
the diameter of a total of 90 pores was measured. Using
2.3. Isolation of rabbit articular chondrocytes the measurement tools in ImageJ software, we accurately
Cartilage cells were isolated from the entire ear cartilage of identified and measured the diameter of each pore.
8-month-old Japanese White rabbits. Cells were obtained Subsequently, we recorded and calculated the diameter
from 10 female animals. After removing the skin tissue data of all pores in each sample group, and conducted
and most of the cartilage membrane, the tissue was cut statistical analysis, including calculating the mean and
into small pieces, with each measuring 1 mm × 1 mm in standard deviation. Finally, we compared the differences in
dimension. These pieces were washed with Dulbecco’s pore diameter between different groups using appropriate
phosphate-buffered saline (DPBS) until clear and then statistical methods.
digested with trypsin for 30 min to remove the remaining
cartilage membrane and connective tissue. Type II 2.5. Dexamethasone release and determination of
collagenase was dissolved in DMEM at a concentration encapsulation efficiency and drug loading efficiency
of 0.2% w/v, and then the treated cartilage pieces were Four groups of MPs, each consisting of three batches of MPs
prepared, totaling 12 samples of 10 mg each, were weighed
processed on a shaker at 37°C for 6–8 h. The digested tissue and transferred into Eppendorf tubes. To each tube, 1 mL
fluid was filtered through a 70 μm mesh, and the cells were of DPBS solution was added, and all the tubes were placed
collected after centrifugation, counted, and cultured and in a 37°C incubator. During each measurement, all DPBS
expanded in cartilage culture medium containing high- solution was aspirated, and 1 mL of fresh PBS solution
glucose DMEM + 10% FBS + 1% PSN at 37°C in a 5% CO was added to replace it. The concentration of DEX in the
2
and 95% humid environment. The second-generation cells supernatant was determined by high-performance liquid
were collected for subsequent experiments. chromatography (HPLC) analysis.
2.4. Preparation of DEX-loaded PLGA MPs The encapsulation efficiency (EE) and drug loading
In this experiment, MPs were prepared using the W/O/W efficiency (DL) of the MPs were measured directly from
method. Briefly, 200 mg of PLGA was dissolved in 8 mL of any one of the batches in each group. The EE and DL were
dichloromethane. DEX in different volumes (15, 30, and 60 calculated using Equations (I) and (II).
μL) was separately dissolved in 150 μL of dimethyl sulfoxide
(DMSO) until completely dissolved, giving rise to solutions
at different concentrations (0.1, 0.2, and 0.4 mg/mL, Wloaded drug
respectively). The DEX-DMSO solution was then added to EE% = × 100 % (I)
the PLGA-DMSO solution to prepare the oil phase. Wfeed drug
In this experiment, 2.5 mL of 1% w/v ammonium
bicarbonate (NH HCO ) solution was utilized as the inner DL% = Wloaded drug × 100 %
4
3
aqueous phase. The prepared inner aqueous phase solution Wpolymer and loaded drug (II)
was gradually added dropwise into the oil phase at a rate of
2.5 mL/min using a syringe pump while stirring with a high- where W is weight.
speed disperser, resulting in the formation of the primary
emulsion. Subsequently, the primary emulsion was slowly 2.6. Preparation of bioink and in vivo
poured into a 0.1% PVA solution (300 mL) while stirring tissue-engineering cartilage culture
at 800 rpm for 3 h to obtain the secondary emulsion. The Cartilage from the upper 1/3 portion of the right ear of
MPs were gathered following the completion of stirring. The Japanese White rabbits was digested with collagenase to
collected MPs were washed three times with deionized water isolate chondrocytes, which were subsequently cultured in
and subsequently hydrolyzed in a 0.1 mol/L NaOH solution vitro until passage 2 (P2) cells were obtained. The cells were
for 20 min. Following hydrolysis, the MPs underwent three then digested to obtain a cell suspension. A control group
7
additional washes with deionized water under the same was prepared by mixing 1 × 10 cells/mL, 10% GelMA w/v,
conditions. Finally, all MPs were filtered to specifically retain and 0.25% LAP w/v.
MPs with a diameter of 75–100 μm, freeze-dried, and stored Four types of MPs (30 mg/mL, w/v), sterilized by
under drying conditions at 4°C for further use. ultraviolet (UV) irradiation, were added to the GelMA

Volume 10 Issue 5 (2024) 386 doi: 10.36922/ijb.3396