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Biomaterials

Biomaterials - Poster Session E

Poster N11 - Development of a Novel Scaffold with Porous Gelatin Microfiber

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Tissue regeneration using cells and scaffolds requires early acquisition of the tissue's original function. It has been reported that cell proliferation and differentiation are promoted by increasing cell activity in scaffolds. Although cell-embedded gel scaffolds can distribute cells uniformly, the lack of nutrient supply into the scaffold reduced cell activity. Porous scaffolds have excellent nutrient supply and diffusion, but poor cell migration due to difficulty in controlling porosity and pore-to-pore continuity. Therefore, a scaffold with excellent connectivity for cell migration and nutrient supply is essential for forming a homogeneous tissue structure.
We hypothesized that the porous microfibers could be used as a scaffold with a mesh structure to promote cell migration and maintain cell activity for a long period. The construction pattern of the microfiber and the distance between the fibers are expected to control the permeability suitable for cell migration, and the porous fibers are expected to maintain cell activity by promoting the retention and supply of the culture medium and body fluids. We focused on gelatin as the scaffold material due to its low-temperature gelation and biocompatibility. A gelatin solution can be gelatinized into fibers by passing it through a cooled microtubule, but continuous spinning requires appropriate cooling conditions. The gel fibers were lyophilized to make them porous, and the effects of gelatin concentration on the porous structure formed in the microfibers needed to be clarified. The purpose of this study was to develop a novel scaffold with porous gelatin microfibers to promote tissue formation.

Materials and

Methods:  The developed spinning apparatus consisted of a stepper motor to dispense the gelatin solution from a syringe, the warming section to maintain the gelatin solution at 25°C, the nozzle equipped with two cooling sections using piezoelectric elements, and the three-axis automatic stage. The nozzle inner diameter was 300 µm, a dispensing rate was 50.3 µL/min and spinning time was 30 minutes. Gelatin powder (Nagase Chemtex) was dissolved in distilled water to prepare 4, 5 and 6 w/v% gelatin solutions. The optimal cooling temperatures for continuous gel fiber formation were determined by setting the first cooling section to 12, 16, 20, and 24 °C, and the second cooling section to 12, 16, 20, and 24 °C. Gelatin gel fibers were frozen at -80°C for 24 hours, and then lyophilized for 24 hours to make them porous. The porous gelatin microfibers were thermally cross-linked at 150°C for 48 hours.
The internal structure was analyzed using X-ray CT, and the pore size and porosity were measured using ImageJ. Water absorption of the porous gelatin microfibers was calculated from the weight of the gelatin microfibers before and after immersing in distilled water. Tensile test was performed using a universal testing machine to measure the tensile strength of the gelatin microfibers.
2.5 × 20 mm fiber sheets were prepared by aligning twelve gelatin microfibers. NIH3T3 cells were seeded at 1.0 × 10^4 cells/cm² on the sheet in the polycarbonate chamber and cultured for 24 and 72 hours in DMEM containing 10% FBS and 1% antibiotics.

Results, Conclusions, and Discussions: The temperature ranges of the first and second cooling sections that allowed for continuous gel fiber formation for 30 minutes were 16-20°C and 12-20°C for 4 w/v% gelatin concentration, 20-24°C and 16-20°C for 5 w/v% gelatin concentration, and 24°C and 16-20°C for 6 w/v% gelatin concentration. Under other conditions, the gelatin solution was either dispensed without gelation or not dispensed due to nozzle blockage. The optimal cooling temperatures (first cooling section, second cooling section) minimizing the variation in fiber diameter of the gel fibers for 4, 5, and 6 w/v% gelatin concentration were determined to be (20,12), (20,20) and (24,20), respectively. Fig. 1 shows CT images of the transverse and longitudinal cross section of the lyophilized fibers for 6 w/v% gelatin concentration. Lyophilized gel fibers had a porous structure for all gelatin concentration. The pore size in the fibers for 4, 5 and 6 w/v% gelatin concentration were 44.3±7.4, 28.9±10.3 and 12.3±3.9 µm, respectively. The pore size decreased with increasing gelatin concentration. The porosity in the fibers for 4, 5 and 6 w/v% gelatin concentration were 88.1, 86.4 and 79.8%, respectively. The porosity decreased with increasing gelatin concentration. These results showed that the decrease in pore size caused by the increase in gelatin concentration resulted in the decrease in porosity. Fig. 2 and Fig.3 show the water absorption and tensile strength of the gelatin microfiber at each gelatin concentration. The water absorption increased with decreasing gelatin concentration, whereas the tensile strength decreased with decreasing gelatin concentration. The increase in porosity caused by the decrease in gelatin concentration enhanced water absorption but reduced the tensile strength because of the decrease in apparent cross-sectional area. The fabricated porous gelatin microfibers for all gelatin concentrations exhibited superior cell adhesion and proliferation.
In this study, we developed the spinning apparatus with two cooling sections to fabricate the continuous and uniform gel fibers, and successfully developed the porous gelatin microfibers through a lyophilization process. The porosity and pore size, which varied with gelatin concentration, affected the water absorption and the tensile strength of the porous gelatin microfibers.

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