Alginate–gelatin hydrogel supplemented with platelet concentrates can be used as bioinks for scaffold printing

In the Laboratory of Tissue Engineering and Biomedical Materials (University of Science, VNU-HCM), Mus musculus var. Albino mice (6–8 weeks old) were housed under temperature and light control (12-h light/dark cycle, 23–28 °C). Water and pellets were freely available to the mice. A total of 38 mice were used to collect approximately 50 mL of blood for all experiments. This study was approved by the Animal Care and Use Committee-University of Science (Certificate of Approval No. 580B/KHTN-ACUCUS), University of Science, VNU-HCM, Vietnam.

To prepare PRP, mouse cardiac blood (about 1.5 mL) was collected and centrifuged at 3,500 rpm for 3 min in a tube containing 150 μL of acid citrate dextrose (ACD) (BD Vacutainer). Approximately 600 μL of plasma was extracted immediately after centrifugation, and this volume was centrifuged for 22 min at 3,000 rpm. After centrifugation, approximately 400 μL of the supernatant was discarded. Then, the pellets were pipetted with the remaining solution (200 μL) of the plasma to obtain non-active PRP. The collected PRP was activated by 20 μL of 230 mM CaCl₂ (Sigma-Aldrich) [14]. The PRP supernatant was then collected from the PRP gel pressing.

To create PRF, mouse cardiac blood was centrifuged at 3,500 rpm for 15 min immediately after collection without anticoagulants. The PRF product only exists in a strongly activated gel form (fibrin clot). Erythrocytes were removed, and the fibrin clot was collected in a separate tube. The PRF supernatant obtained from pressing PRF was then collected.

The PRP and PRF supernatants were stored at −20 °C until further use.

Alginate (Sigma-Aldrich), gelatin (Sigma-Aldrich), and CaCl₂ (Sigma-Aldrich) powders were dissolved in deionized water (Sigma-Aldrich), respectively. The AG bioink was prepared by mixing solutions of 10% (w/v) alginate, 10% (w/v) gelatin, and 70 mM CaCl₂ in a ratio of 1:1:0.5 by volume, respectively. The bioinks containing platelet concentrates were AG-PRP (AGP) bioink and AG-PRF (AGF) bioink, which was created by adding PRP and PRF supernatants into the AG bioink (1:10, v/v), respectively. These bioinks were transferred to a 3 mL syringe, and the bubbles in the bioink were removed by centrifuging at 3,000 rpm for 5 min.

The gelation of both AG, AGP, and AGF bioinks was determined by inverting the tubes containing the bioinks. These bioinks were added to the vials and sealed with screw caps. These vials were incubated at room temperature, and at regular intervals, the vials were inverted to check for a sol–gel transition. To check the pH of the bioinks, litmus papers were used. This quick method was carried out in order to determine whether bioinks are suitable for cell survival.

The scaffolds were created using a 3D bioprinter with an extrusion-based design. The Solidworks software was used to model a cube (20 mm × 20 mm × 1 mm). The bioprinter (Bio X, Cellink) was loaded with a 3-mL syringe containing AG, AGP, and AGF bioinks. The following settings were chosen for 3D bioprinting: a needle with a 250-μm inner diameter, platform room temperature, fill density of 10%, and four layers with a layer thickness of 250 μm. After printing, the AG, AGP, and AGF scaffolds were cross-linked by soaking in 0.1 M calcium chloride (Sigma-Aldrich) for 10 min and then washed three times in 1X PBS (Gibco).

The cytotoxicity of AG, AGP, and AGF scaffolds was assessed using an extract test followed by an MTT (3-[4-C-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich) assay, according to ISO 10993-5 [15]. Both basic medium (DMEM/F12 [Sigma-Aldrich], 1% penicillin/streptomycin [Sigma-Aldrich]), and cell culture medium (DMEM/F12 [Sigma-Aldrich], 10% FBS [Sigma-Aldrich], 1% penicillin/streptomycin [Sigma-Aldrich]) were used to obtain extracts. These two types of extracts were referred to as serum-containing extracts and serum-free extracts, respectively. Scaffolds had a surface area of about 3 cm² and were incubated in 1 mL of these media at 37 °C for 24 h. After incubation, the scaffold extracts were collected. The L929 cell line was cultured in 96-well plates, where each well contained 10⁴ cells until 80% confluency. Then, 100 μL of extracts were applied to each of the 96-well plates, which were then incubated for 24 h at 37 °C in a humidified 5% CO₂. The media (the cell culture medium or the basic medium) and 20% DMSO were used as negative and positive controls, respectively. After discarding the medium, each well was filled with 100 μL of 0.5 mg/mL MTT solution (Sigma-Aldrich) and incubated for 4 h to form formazan crystals. After carefully discarding the liquids in each well, 100 μL DMSO: Ethanol (Sigma-Aldrich) (1:1, v/v) was pipetted into each well, giving the cell culture plate a uniform color. A microplate reader (Biochrom EZ Read) was used to measure the absorbance at a wavelength of 570 nm. The same experiment was performed with a serum-free medium for extract collection. The relative growth rate (RGR) was calculated as The relative growth rate (RGR)%=Absorbance of treated wellsAbsorbance of cell control×100 {\rm{The}}\;{\rm{relative}}\;{\rm{growth}}\;{\rm{rate}}\;({\rm{RGR}}){\rm{ }}\% = {{{\rm{Absorbance}}\;{\rm{of}}\;{\rm{treated}}\;{\rm{wells}}} \over {{\rm{Absorbance}}\;{\rm{of}}\;{\rm{cell}}\;{\rm{control}}}} \times 100

The release of GFs from AG, AGP, and AGF scaffolds was assessed over 12 d. Scaffold extracts were collected at selected time points, including days 1, 4, 8, and 12, and stored at −20 °C until further use. The scaffolds were incubated in DMEM-F12 medium at 37 °C for 24 h to produce scaffold extracts. After 24 h, the solution was collected (day 1). Next, fresh DMEM-F12 medium was added, and incubation was maintained until day 4 when the supernatant was collected (day 4). The same procedure covered the following two-time intervals, including days 8 and 12. PRP and PRF supernatants were used as the controls. The total GF release was measured using ELISA kits specific for platelet-derived growth factor-AB (PDGF-AB, RAB1857-1KT, Sigma-Aldrich), and vascular endothelial growth factor (VEGF, RAB0509-1KT, Sigma-Aldrich).

IBM SPSS Statistics V22.0 software (IBM Corporation) was used in this study. The data were presented as Mean ± SD and two-tailed Student's t-tests, one sample t-test, and ANOVA test were performed to assess statistically significant between groups (P < 0.05).

The gelation of a bioink determines its sol–gel transition. The time it takes for a solution to turn into a gel is known as the gelation time. It is typically optimized to adjust for the rheological properties of bioinks. Figure 1 shows the results of a survey conducted by the tube inversion method. We found that all of the bioinks, including AG, AGP, and AGF maintained their shape after the tubes were inverted. Gelation was presented immediately after blending bioink components, and this state was maintained for up to 15 min (enough time to complete print operations after that). The sol–gel transition times of three bioinks were not statistically different. On the contrary, to ensure the compatibility of composite materials from these bioinks, the bioinks’ pH needs to be controlled. By comparing with the color of the standard pH scale on litmus paper, the pH values of the AG bioink, AGP bioink, and AGF bioink all had a neutral pH of around 7 (Figure 2).

Figure 1.

Figure 2.

GF release is a required property of the scaffolds that plays an essential role in providing biologically active substances that promote tissue regeneration. This study demonstrated that the scaffolds that were compounded with a supernatant of 10% PRP or PRF could secrete PDGF-AB and VEGF for 12 d. However, there was a difference in the GF release between the two types of scaffolds (Figure 3).

Figure 3.

On day 1, the PDGF-AB concentrations were the highest in all scaffolds for PDGF-AB release. At AGF scaffolds, this concentration was 71.67 ± 2.60 pg/mL, which remained constant for 8 d (P > 0.05) and decreased on day 12 (47.92 ± 4.73 pg/mL) (P < 0.05). However, not for AGP scaffolds, which decreased over time throughout the survey. On the contrary, PDGF-AB concentrations from PRP and PRF, which were added to AG hydrogel (10% in final hydrogels), were different. In PDGF-AB, concentration in the added-PRP was 89.37 ± 0.79 pg/mL, which was higher than in the added-PRF (61.22 ± 0.79 pg/mL) (P < 0.05). It was observed that the concentration of PDGF-AB secreted at AGF scaffolds was equivalent to that at 10% PRF added. It was different from AGP scaffolds when this GF was secreted at a lower concentration of 10% of the original PRP.

In contrast to PDGF-AB release, VEGF is stably secreted at AGP scaffolds, and the secretion of this GF tends to decrease rapidly over time at AGF scaffolds (Figure 3). Specifically, the VEGF concentrations from AGP scaffolds on days 1, 4, 8, and 12 were 3.33 ± 0.57 pg/mL, 2.79 ± 0.24 pg/mL, 4.03 ± 0.16 pg/mL, and 3.01 ± 0.19 pg/mL, respectively, and there were no statistically significant differences between them (P > 0.05). However, these concentrations were lower than the concentration of VEGF from PRP, which was added to AG hydrogel at a 10% concentration. Unlike the AGP scaffolds, the concentration of VEGF secreted by the AGF scaffolds on day 1 was 13.98 ± 0.40 pg/mL, which was higher than by 10% of the PRF added to the original hydrogels (8.62 ± 0.88 pg/mL) (P < 0.05). The concentration of this GF secreted from AGF scaffolds on day 8 of the investigation was equivalent to that from AGP scaffolds (P-value > 0.05). However, by day 12, VEGF secretion was not observed (Figure 3).

The effect of AG, AGP, and AGF scaffolds on cell viability was evaluated through an MTT assay. Results showed that, in the serum-free extracts, the RGR of cells was highest in the AGF group, at 173.69 ± 15.48 (P < 0.05). This rate was found in the AG and AGP groups to be 130.36 ± 3.58 and 154.69 ± 22.21, respectively. Moreover, the cell growth in these three groups was higher than in the negative control group (P < 0.05). With the impact of 20% DMSO, the positive control had the lowest rate of cell growth, reaching 1.81 ± 0.78 (P < 0.05) (Figure 4).

Figure 4.

On the contrary, in the serum-free extracts, the cell growth rates in the AG, AGP, and AGF groups were lower than in the negative control. These rates were 74.35 ± 0.16, 75.17 ± 9.83, and 86.75 ± 6.63, respectively. However, the cell growth rates in the PRP and PRF supplementation groups were higher than the positive control (P < 0.05). It was still over 70% (Figure 5). According to ISO 10993-5, if the RGR for the concentrations of the extracts is ≥70% of the positive control group, then the material shall be considered to be non-cytotoxic.

Figure 5.