Characterization of Porous Fibrin Scaffolds with Tunable Anisotropic Features

Introduction: Volumetric muscle loss (VML) impacts 65.8 million Americans annually, including a disproportionate number of wounded warriors injured in combat. VML is caused when significant skeletal muscle loss damages the biophysical and biochemical signaling cues that direct functional muscle regeneration. Tissue engineering strategies that use biomimetic scaffolds offer a promising solution for treating VML by providing anisotropic topographic features to guide myotube alignment which is important to produce uniaxial contractile forces. Recently, our lab created anisotropic fibrin scaffolds using directional lyophilization, where anisotropic scaffold architectures are tuned by altering fabrication parameters (Samolyk et al., Tissue Eng Part C, 2024). We showed that scaffolds fabricated with lower freezing temperatures and higher fibrin concentrations result in maximized myoblast orientation. Here, we aim to further characterize the properties of anisotropic scaffolds under physiologic conditions. The aim of this study is to determine how the fabrication parameters impact the hydration, porosity, and degradation properties of the scaffolds to predict how they will perform after implantation.

Materials and

Methods: Several fabrication parameters were assessed for the scaffolds, including fibrin concentrations of 10 and 20 mg/mL as well as freezing temperatures of -40, -80, and -195C. To assess hydrated scaffold architecture, scaffolds were soaked in PBS for 15 min, fixed, and paraffin embedded. Scaffold sections (8 μm thick) were cut in a plane parallel to the freezing direction, mounted, stained with eosin, and imaged in brightfield. Feature size analyses were performed by manually measuring the distance between parallel struts within the scaffolds using ImageJ. To characterize the porosity of the scaffolds, ethanol immersion was used on rectangular sections. Initial weight, length, width, and height were measured, then samples were submerged in 100% ethanol for 2 min, followed by a second weight measurement used to calculate percent porosity. To assess changes in scaffold morphology due to hydration, scaffold dimensions were quantified when dry and hydrated. Scaffolds were sectioned into 1 mm thick samples, and then 2 mm diameter biopsy punches were used to create samples. Brightfield images of the planar surfaces of scaffolds were taken before and after hydration in PBS for 1 h, then analyzed with ImageJ to quantify changes in scaffold area. To assess proteolytic degradation, scaffolds were incubated in 0.2 mL of 0.5 U/mL human plasmin in PBS at 37C. Scaffolds were imaged at 0, 3, 6, 9, 24, 48, and 72 hours after the addition of plasmin. Each timepoint was analyzed with ImageJ to quantify the reduction in scaffold surface area.

Results, Conclusions, and Discussions: To quantify the effect of hydration on scaffold morphology, we measured changes in strut width as well as bulk changes in dimensional surface area. Average strut widths for hydrated directional scaffolds ranged from 3.42-21.91 μm depending on the experimental condition. Interestingly, although the strut widths were smaller on hydrated scaffolds compared to dry scaffolds, the same trends were followed. The -40C, 10 mg/mL scaffolds showed significantly increased strut width compared with all other scaffold conditions. When observing the bulk dimensional changes of the material, the average shrinkage ranged from 24.20-43.98%. The -40C, 10mg/mL scaffolds shrank significantly more than both -195C scaffold groups. There was also a general trend, where scaffolds with smaller strut widths displayed less shrinkage than those with larger strut widths. Scaffold porosity was quantified using ethanol immersion. Average porosities ranged from 79.00-94.16%; however, porosity was not significantly different for any of the scaffold groups. Proteolytic degradation of scaffolds was evaluated over 4 d, periodically measuring the decrease in initial area of samples. For all the scaffold groups, there was a sharp decrease in area after the first 3 h in plasmin, followed by a more gradual decrease in area. In contrast, the no plasmin control maintained a relatively flat curve, with no sharp decrease in area. None of the scaffold groups were significantly different from one another at each time point. However, the -80C, 10 mg/mL and the -195C, 10 mg/mL scaffolds did display significant decreases in area compared with no plasmin scaffold controls at 24, 48, and 72 hours.
Here, we describe the creation of a novel porous fibrin scaffold with anisotropic microarchitectural features. Structural and morphological evaluations of our scaffolds were used to characterize these constructs under physiological conditions. We showed that the scaffolds all exhibited a degree of shrinkage upon hydration, which has been observed in other porous biologic scaffolds. We also demonstrated that porosity and proteolytic degradation are not altered by the fabrication parameters we explored. Future work will focus on myotube formation on these scaffolds.

Acknowledgements (Optional): This work was funded in part by AHA Institutional Research Enhancement Award 953260 and NIH 1R15AR080988.