Preparation of GelMA bioink. The purchased GelMA (EFL-GM-100, purity > 99.9%) powder was removed from a -80°C refrigerator and then dissolved with deionized water to the concentration of 15% w/v. Photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, purity > 99.8%) was then added into the solution to reach a concentration of 0.5% w/v. The resulting solution was sufficiently dissolved using a 50°C water bath. Then, the light absorber Tartrazine (purity > 95%) was added into the GelMA bioink to reach a concentration of 0.03% w/v. The prepared GelMA bioink was then stored in a 4°C refrigerator.
Printing of pcGelMAs by PBP. The pcGelMAs were printed by a desktop PBP printer (EFL-BP8600). First, the printer was powered on and the preheating temperature required for printing the material was set. Both the deposition platform and bioink pool of the printer were then removed. The inside of the bioink pool and the formation area of the deposition platform were each wiped down with a non-woven cloth dipped in 75% alcohol. They were then dried and placed back on the printer. The designed 3D digital model was exported to the ‘.STL’ file format and imported into the printer's host computer software to adjust its size, position, and layout. The light intensity, curing time, and printing temperature were set. After the printer had preheated, the prepared bioink was aspirated a certain amount with a pipette gun and then added to the bioink pool. The amount of bioink added was evaluated according to the actual volume of the structure to be printed. The protective cover was placed and then ‘Start Printing’ was selected. After printing, the printing deposition platform was carefully removed. The hydrogel structure of the printing molding was also carefully removed with a spatula and then immediately placed into the isotonic solvent to wash away the remaining uncrossed bioink on its surface. The washed hydrogel holder was removed from the isotonic aqueous solution, transferred to 75% medical alcohol for immersion, and then placed in a 4°C refrigerator for long-term storage.
Lyophilization treatment of the pcGelMAs. The photocrosslinked GelMA hydrogels that had reached swelling equilibrium were transferred into well plates. The well plates were then placed in a -80°C refrigerator for 1 hour to fix the hydrogel morphology, during which time, the vacuum drier was turned on and the cooling temperature was set to -80°C. Then, the well plates containing the hydrogels were transferred into the vacuum drier and the pump was turned on to remove all the gas inside. After 24 hours, the freeze-dried and vacuum-sealed GelMA hydrogels were stored.
Dampening treatment of the fd-pcGelMAs. The heater temperature of the dampening treatment device was set to 50°C and the treatment conditions were 25°C and 95%. The fd-pcGelMAs were transferred to a 24-well plate, which was followed by being placed on the metal netting of the dampening treatment device. The treated fd-pcGelMAs (soggy-pcGelMAs) were removed from the device at different time points and restored in a plastic bag using a food vacuum packaging machine.
Volume measurement of the soggy-pcGelMAs. From the soggy-GelMA volume observations made during the dampening treatment, soggy-GelMA cuboids fabricated in the same batch were treated by the dampening treatment device simultaneously before being removed for volume analysis at 0.5, 1, 2, 4, 6, and 22 hours (n = 3). Random deformation would occur during both the freeze-drying process of the photocrosslinked GelMA hydrogels and the dampening process of the fd-GelMA. Therefore, obtaining direct measurements with the use of a ruler was impossible. Here, ImageJ was applied to analyze the upper area \({S}_{upper}\left(t\right)\) (regarded as a standard square) of the samples according to photos obtained by a camera (Nikon SLR) and the use of a ruler. Importantly, the molecular network inside the photocrosslinked GelMA hydrogel was roughly isotropic and the sizes in three orthogonal directions could change in equal proportions \(\epsilon\) during the dampening process. Therefore, the volume of the soggy-pcGelMA \({V}_{soggy-GelMA}\left(t\right)\) at a time point \(t\) could be calculated as below:
$${V}_{soggy-pcGelMA}\left(t\right)={V}_{design}\times {\left(\frac{{S}_{upper}\left(t\right)}{{S}_{upper-design}}\right)}^{\frac{3}{2}}$$
where \({V}_{design}\) and \({S}_{upper-design}\) were the volume and the upper area of the designed photocrosslinked GelMA hydrogel (mold size), respectively.
Water content measurement of the soggy-GelMA. fd-GelMA cuboids fabricated in the same batch were treated by the dampening treatment device simultaneously and then removed for water content analysis at 0.5, 1, 2, 4, 6, and 22 hours (n = 3). The fd-GelMA and soggy-GelMA were lightly wiped with a non-woven fabric to remove the water on their surface. Then, the masses of the fd-GelMA \({m}_{fd-GelMA}\) and soggy-GelMA \({m}_{soggy-GelMA}\left(t\right)\) were respectively measured with an electronic balance. The water content of the soggy-GelMA \({\psi }_{soggy-GelMA}\left(t\right)\) at a time point \(t\) could be calculated as below:
$${\psi }_{soggy-GelMA}\left(t\right)=\frac{{m}_{soggy-GelMA}\left(t\right)-{m}_{fd-GelMA}}{{m}_{fd-GelMA}}\times 100\%$$
Compression testing of the soggy-pcGelMAs. Bioink was printed by a PBP printer into φ4 mm × 4 mm and then immersed in PBS for 24 h to reach swelling equilibrium. Then, L&D treatment was carried out on the pcGelMAs (n = 3). A universal testing machine (UTM-2102) was used to test the compression modulus using a 100 N mechanical sensor, a bar compression test module, an inlet force of 0.001 N, and a compression test rate of 1 mm/min. The device automatically recorded the force-displacement curve. After the test, the "force-displacement" curve data was exported, underwent standardization processing in Excel, and was converted into a "stress-strain" curve to eliminate the influence of different dimensions on the test results.
Surface hydrophilia of the soggy-pcGelMAs. The hydrophilia of the soggy-pcGelMAs (n = 3) was determined by a contact angle surface tension measuring instrument (Dropmeter 100P). The soggy-pcGelMAs were placed on the sample stage of the surface contact angle tester. Then, a 4 µL droplet (\({V}_{\text{d}\text{r}\text{o}\text{p}\text{l}\text{e}\text{t}}\)) was dropped on the surface of the samples (\({t}_{0}\)). A high-speed digital camera was utilized to take pictures; the shooting time interval of each photo was set to 0.3 s. The obtained photos were analyzed frame by frame and used to measure the size and change in the contact angle of the droplets in each photo. The time point at which the droplet had completely infiltrated was recorded as \({t}_{i}\). The average infiltration rate of the droplet can be expressed by the following formula:
$${\stackrel{-}{v}}_{\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}}=\frac{{V}_{\text{d}\text{r}\text{o}\text{p}\text{l}\text{e}\text{t}}}{{t}_{i}-{t}_{0}}$$
PBP printing of the BioBullets. BioBullets featuring grids were designed with Solidworks software. The pore size was set to 600 µm. To compensate for the effect of the crosslinking depth on the side pore size, the side pore height was set to 1000 µm. The model was imported into slicing software. The layer height of the slice was set to 100 µm. The printed pcGelMAs were then carefully scraped off the deposition platform with a razor blade and subsequently washed in PBS to remove the residual bioink, which was followed by immersing the pcGelMAs in PBS for 24 h to reach swelling equilibrium. The resulting pcGelMAs then underwent L&D treatment. The dampening process lasted for 2 hours.
SEM morphology of the BioBullets. The morphology of the pore network in the BioBullets was observed with a scanning electronic microscope (SEM). The samples were frozen with liquid nitrogen and transferred to a vacuum freeze dryer at -80°C for 24 h. The resulting freeze-dried samples were then torn along the cross-section and coated with a sputtering coater.
Degradation of the BioBullets. The BioBullets were re-swelled in deionized water for 24 hours and then placed in a 1.5 mL EP tubes in a 37°C, 5% CO2 incubator. Then, 1 mL of PBS containing 2 U/mL type II collagenase was added to each tube. After 0, 1, 2, and 4 h of degradation, the solution in the EP tube was removed and the tubes were transferred to a -80°C refrigerator for storage. After removing all the degraded samples, those remaining were lyophilized by a vacuum freeze dryer for 24 hours. The final weights of the freeze-dried samples were then respectively weighed.
ADSC seeding on the BioBullets. The BioBullets were placed into a sterile 96-well plate and irradiated with UV light for 30 minutes on a sterile ultra-clean bench. ADSCs were then digested in culture dishes with 0.25% EDTA-trypsin and resuspended with a small amount of medium to reach a final cell concentration of approximately 5 × 106 /mL. Then, 100 µL of the cell suspension was pipetted gently onto the center of the surface of the BioBullets. The cell suspension then underwent rapid absorption by the BioBullets and the volume expanded. The inoculated scaffolds were placed on a clean table for 30 minutes before being transferred to a 37°C, 5% CO2 incubator for 4 hours of incubation. After 4 hours, 100 µL of cell culture medium was slowly added to each well to ensure that the liquid level did not cover the scaffold. This was then transferred to a CO2 incubator to continue the incubation.
Modeling of adipose tissue defects and minimally invasive injection using BioGun. 8-week-old female mice from the ICR (Institute of Cancer Research) were anesthetized with 1% pentobarbital before their side hair was shaved using a body hair machine. Then, the surgical area was depilated with depilatory cream and thoroughly cleaned with 75% alcohol. The subcutaneous fat in the inguinal region of the mice was selected from the surgical area. The inguinal fat of each mouse was located, a piece was cut off with sterile scissors, and the incision was sutured. For the minimally invasive injection process, each part of the BioGun was thoroughly wiped with 75% alcohol and UV-irradiated overnight on a sterile bench. Then, the BioBullet was compressed, loaded into a glass cartridge case, and pressed into the magazine. A certain amount of phosphate-buffered saline (PBS) was drawn with a 2 mL syringe and then assembled according to the operating procedure. The minimally invasive injection of the BioBullet was completed under the guidance of small animal B-ultrasound. The metal tip of the BioGun was placed into the skin of the mouse to locate the adipose tissue. Under real-time display using B-ultrasound, the metal gun barrel has a strong white echo with a sound shadow. A pipette tip was inserted deeply into the defect site of the adipose tissue and the piston was pressed to push out the compressed GelMA scaffold. The water injection trigger was squeezed to inject a certain amount of PBS in situ.
Histological analysis. BioBullets within newborn tissues were removed on the 14th, 28th, and 40th days after their injection, respectively. They were then dehydrated, embedded, sectioned, and stained with H&E to show the ingrowth of cells around the scaffold. In order to more clearly show the distribution of the newly grown cells inside of the BioBullets, during the sectioning process, sections were sectioned at a thickness of 6 µm, and those near the BioBullet-containing tissue were selected after staining. In order to show the ingrowth trend of adipocytes within 40 days of scaffold repair, ImageJ software was used to quantify the newly formed vacuolar adipose tissue in the slices.
CD31 immunohistochemistry. Dewaxing and hydration were conducted with the following at room temperature, in chronological order: xylene No. 1 for 10 minutes, xylene No. 2 for 8 minutes, xylene No. 3 for 8 minutes, absolute ethanol for 5 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol for 3 minutes, ethanol for 3 min, distilled water for 3 minutes × 3 times, and PBS for 3 minutes × 3 times. For hot antigen retrieval, a new slide rack was used to replace the old one, and the hydrated slides were placed. Antigen retrieval solution was prepared using a packet of sodium citrate powder to prepare 2 L of PBS solution. This was then heated and boiled in an iron lunch box or pot. The temperature was controlled at 96–98°C. The slices were then placed in a hot pan for 15 minutes before being naturally cooled to room temperature. The slices were then rinsed with PBS for 5 minutes × 2 times, followed by distilled water for 3 minutes × 2 times. The tissue was removed, shaken to dry, and absorbed of water droplets using absorbent paper. A histochemical stroke circle was used to surround the tissue. Inactivation of endogenous enzyme activity was conducted as follows: the slices were placed in a wet box, added with a small amount of distilled water, and then added with 3% hydrogen peroxide dropwise (one drop or 50 µL of A in the secondary antibody kit; see the instructions for the secondary antibody for details). This was incubated for 10 minutes at room temperature. The slices were then washed with PBS for 3 minutes × 3 times, followed by distilled water for 3 minutes. To block non-specific sites: the water was spun dry, the water beads were absorbed, goat serum blocking solution was added (a drop of secondary antibody kit B or 50 µL), and the slides were placed in a humid box and left at room temperature for 10 minutes. Then, the blocking solution was shaken off, the primary antibody was added dropwise to cover the tissue, and the tissue was placed in a humid chamber at 4°C overnight. For the concentration of primary antibody, refer to the instruction manual of the antibody; antibodies were diluted in advance, divided into packaging, and then stored in a refrigerator at -20°C. The dose of the primary antibody differed for each tablet depending on the size of the tissue. Each sample of cancer tissue occupied approximately 20 µL in volume. The next day, the wet box was removed from the 4°C refrigerator, rewarmed at room temperature for 30 minutes, washed with PBS for 3 minutes × 3 times, and then washed with distilled water for 2 minutes. The water was then drained, the water droplets were absorbed, and either one drop or 50 µL of the secondary antibody was added (Secondary Antibody Kit C) dropwise before incubation was carried out at room temperature for 10 minutes. This was followed by washing with PBS for 3 minutes × 3 times, followed by distilled water for 3 minutes. Then, one drop or 50 µL of streptavidin-peroxidase solution (secondary antibody kit D) was added to each slide before they were incubated at room temperature for 10 minutes. They were then washed with PBS for 3 minutes × 3 times, followed by rinsing with distilled water for 3 minutes. Either two drops or 100 µL of DAB solution was then added to each slide before they were observed under a microscope for 3–10 minutes. The dyeing was terminated when appropriate before being rinsed off with tap water. Hematoxylin counterstaining was conducted for 1–2 minutes. When the nuclei turned blue, the dyeing process was stopped and the slide was rinsed with tap water or PBS (reverse blue with 0.5% ammonia). The slides were then placed in 1% hydrochloric acid alcohol for 3 seconds, followed by rinsing with tap water for 3 min. Slide dehydration was conducted as follows, in chronological order: 70% alcohol for 1 minute, 80% alcohol for 1 minute, 95% alcohol for 2 minutes, absolute ethanol for 4 minutes, xylene 1 for 3 minutes, and xylene 2 for 3 minutes. After drying the slides, add an appropriate amount of neutral gum was added to cover the slides. The slides were then covered with a cover glass to remove any air bubbles. They were finally left to dry before photos were captured under a microscope.