Static axial stretching enhances the mechanical properties and cellular responses of fibrin microthreads

Abstract

Fibrin microthreads are a platform technology that can be used for a variety of applications, and therefore the mechanical requirements of these microthreads differ for each tissue or device application. To develop biopolymer microthreads with tunable mechanical properties, we analyzed fibrin microthread processing conditions to strengthen the scaffold materials without the use of exogenous crosslinking agents. Fibrin microthreads were extruded, dried, rehydrated, and static axially stretched 0-200% of their original lengths; then the mechanical and structural properties of the microthreads were assessed. Stretching significantly increased the tensile strength of microthreads threefold, yielding scaffolds with tensile strengths and stiffnesses that equaled or exceeded values reported previously for carbodiimide crosslinked threads without affecting intrinsic material properties such as strain hardening or Poisson's ratio. Interestingly, these stretching conditions did not affect the rate of proteolytic degradation of the threads. The swelling ratios of stretched microthreads decreased, and scanning electron micrographs showed increases in grooved topography with increased stretch, suggesting that stretching may increase the fibrillar alignment of fibrin fibrils. The average cell alignment with respect to the longitudinal axis of the microthreads increased twofold with increased stretch, further supporting the hypothesis that stretching microthreads increases the alignment of fibrin fibrils on the surfaces of the scaffolds. Together, these data suggest that stretching fibrin microthreads generates stronger materials without affecting their proteolytic stability, making stretched microthreads ideal for implantable scaffolds that require short degradation times and large initial loading properties. Further modifications to stretched microthreads, such as carbodiimide crosslinking, could generate microthreads to direct cell orientation and align tissue deposition, with additional resistance to degradation for use as a long-term scaffold for tissue regeneration.

Biomimetic scaffolds for regeneration of volumetric muscle loss in skeletal muscle injuries

“…While we observed a reduction in the deposition of scar tissue 3–4 months post-injury, histological analyses of tissue harvested at early time points suggested that the microthreads were largely degraded within 2 weeks of implantation and many of the regenerated myofibers in the wound site at later time points exhibited some degree of misalignment with respect to the native muscle tissue [91]. We developed a method to attenuate the degradation rate of fibrin microthreads using carbodiimide crosslinking chemistry to enhance the in vitro persistence of these scaffolds [219]. Interestingly, discrete structural and mechanical properties were developed by modifying the pH environment of the carbodiimide crosslinking reaction, suggesting that the efficiency of carbodiimide crosslinking can be regulated both through controlling the pH environment as well as the total crosslinking time [219].…”

“…We developed a method to attenuate the degradation rate of fibrin microthreads using carbodiimide crosslinking chemistry to enhance the in vitro persistence of these scaffolds [219]. Interestingly, discrete structural and mechanical properties were developed by modifying the pH environment of the carbodiimide crosslinking reaction, suggesting that the efficiency of carbodiimide crosslinking can be regulated both through controlling the pH environment as well as the total crosslinking time [219]. …”

“…Applying static axial stretching techniques to natural polymers may facilitate the alignment of protein fibrils within the bulk scaffold, which will direct uniaxial alignment of cells. However to date, these materials have not yet been applied to VML injuries [219, 221]. Fibrin microthreads have been shown to direct aligned muscle regeneration (Figure 3C), but these microthreads degrade rapidly in vivo [91].…”

Designing Biopolymer Microthreads for Tissue Engineering and Regenerative Medicine

“…[2] When microthreads were stretched between 0 –200% of their initial lengths, threads stretched 150% of their initial length exhibited a three-fold increase in tensile strength. [2] Additionally, stretched threads increased the alignment of C2C12 myoblasts seeded on the surface of threads, with respect to untreated microthread controls. [2] Together, these findings show that static axial stretching significantly enhanced mechanical properties and the cellular responses of fibrin microthreads.…”

“…Biopolymer microthreads are discrete, fibrous materials, generated from ECM and naturally derived proteins such as fibrin [2–4], silk [5, 6], collagen [7, 8], chitosan, and alginate [9–11]. The morphological and biochemical properties of the these microthreads are comparable to native fibrous structures, and they can be precisely engineered into hierarchically ordered, tissue-specific scaffolds with morphological, mechanical and biochemical cues to promote cell mediated tissue regeneration (Figure 1).…”