RESUMO
Aim: The pleiotropic effect of fibroblast growth factor 2 (FGF2) on promoting myogenesis, angiogenesis, and innervation makes it an ideal growth factor for treating volumetric muscle loss (VML) injuries. While an initial delivery of FGF2 has demonstrated enhanced regenerative potential, the sustained delivery of FGF2 from scaffolds with robust structural properties as well as biophysical and biochemical signaling cues has yet to be explored for treating VML. The goal of this study is to develop an instructive fibrin microthread scaffold with intrinsic topographic alignment cues as well as regenerative signaling cues and a physiologically relevant, sustained release of FGF2 to direct myogenesis and ultimately enhance functional muscle regeneration. Methods: Heparin was passively adsorbed or carbodiimide-conjugated to microthreads, creating a biomimetic binding strategy, mimicking FGF2 sequestration in the extracellular matrix (ECM). It was also evaluated whether FGF2 incorporated into fibrin microthreads would yield sustained release. It was hypothesized that heparin-conjugated and co-incorporated (co-inc) fibrin microthreads would facilitate sustained release of FGF2 from the scaffold and enhance in vitro myoblast proliferation and outgrowth. Results: Toluidine blue staining and Fourier transform infrared spectroscopy confirmed that carbodiimide-conjugated heparin bound to fibrin microthreads in a dose-dependent manner. Release kinetics revealed that heparin-conjugated fibrin microthreads exhibited sustained release of FGF2 over a period of one week. An in vitro assay demonstrated that FGF2 released from microthreads remained bioactive, stimulating myoblast proliferation over four days. Finally, a cellular outgrowth assay suggests that FGF2 promotes increased outgrowth onto microthreads. Conclusions: It was anticipated that the combined effects of fibrin microthread structural properties, topographic alignment cues, and FGF2 release profiles will facilitate the fabrication of a biomimetic scaffold that enhances the regeneration of functional muscle tissue for the treatment of VML injuries.
RESUMO
Functional regeneration of anisotropically aligned tissues such as ligaments, microvascular networks, myocardium, or skeletal muscle requires a temporal and spatial series of biochemical and biophysical cues to direct cell functions that promote native tissue regeneration. When these cues are lost during traumatic injuries such as volumetric muscle loss (VML), scar formation occurs, limiting the regenerative capacity of the tissue. Currently, autologous tissue transfer is the gold standard for treating injuries such as VML but can result in adverse outcomes including graft failure, donor site morbidity, and excessive scarring. Tissue-engineered scaffolds composed of biomaterials, cells, or both have been investigated to promote functional tissue regeneration but are still limited by inadequate tissue ingrowth. These scaffolds should provide precisely tuned topographies and stiffnesses using proregenerative materials to encourage tissue-specific functions such as myoblast orientation, followed by aligned myotube formation and recovery of functional contraction. In this study, we describe the design and characterization of novel porous fibrin scaffolds with anisotropic microarchitectural features that recapitulate the native tissue microenvironment and offer a promising approach for regeneration of aligned tissues. We used directional freeze-casting with varied fibrin concentrations and freezing temperatures to produce scaffolds with tunable degrees of anisotropy and strut widths. Nanoindentation analyses showed that the moduli of our fibrin scaffolds varied as a function of fibrin concentration and were consistent with native skeletal muscle tissue. Quantitative morphometric analyses of myoblast cytoskeletons on scaffold microarchitectures demonstrated enhanced cell alignment as a function of microarchitectural morphology. The ability to precisely control the anisotropic features of fibrin scaffolds promises to provide a powerful tool for directing aligned tissue ingrowth and enhance functional regeneration of tissues such as skeletal muscle.