On the role of intrinsic and extrinsic forces in early cardiac S-looping.

BACKGROUND: Looping is a crucial phase during heart development when the initially straight heart tube is transformed into a shape that more closely resembles the mature heart. Although the genetic and biochemical pathways of cardiac looping have been well studied, the biophysical mechanisms that actually effect the looping process remain poorly understood. Using a combined experimental (chick embryo) and computational (finite element modeling) approach, we study the forces driving early s-looping when the primitive ventricle moves to its definitive position inferior to the common atrium. RESULTS: New results from our study indicate that the primitive heart has no intrinsic ability to form an s-loop and that extrinsic forces are necessary to effect early s-looping. They support previous studies that established an important role for cervical flexure in causing early cardiac s-looping. Our results also show that forces applied by the splanchnopleure cannot be ignored during early s-looping and shed light on the role of cardiac jelly. Using available experimental data and computer modeling, we successfully developed and tested a hypothesis for the force mechanisms driving s-loop formation. CONCLUSIONS: Forces external to the primitive heart tube are necessary in the later stages of cardiac looping. Experimental and model results support our proposed hypothesis for forces driving early s-looping.

A biomimetic approach to modulating the sustained release of fibroblast growth factor 2 from fibrin microthread scaffolds.

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.

Etching anisotropic surface topography onto fibrin microthread scaffolds for guiding myoblast alignment.

To regenerate functional muscle tissue, engineered scaffolds should impart topographical features to induce myoblast alignment by a phenomenon known as contact guidance. Myoblast alignment is an essential step towards myotube formation, which is guided in vivo by extracellular matrix structure and micron-scale grooves between adjacent muscle fibers. Fibrin microthread scaffolds mimic the morphological architecture of native muscle tissue and have demonstrated promise as an implantable scaffold for treating skeletal muscle injuries. While these scaffolds promote modest myoblast alignment, it is not sufficient to generate highly functional muscle tissue. The goal of this study is to develop and characterize a new method of etching the surface of fibrin microthreads to incorporate aligned, sub-micron grooves that promote myoblast alignment. To generate these topographic features, we placed fibrin microthreads into 2-(N-morpholino)ethane-sulfonic acid (MES) acidic buffer and evaluated the effect of buffer pH on the generation of these features. Surface characterization with atomic force microscopy and scanning electron microscopy indicated the generation of aligned, sub-micron sized grooves on microthreads in MES buffer with pH 5.0. Microthreads etched with surface features had tensile mechanical properties comparable to controls, indicating that the surface treatment does not inhibit scaffold bulk properties. Our data demonstrate that etching threads in MES buffer with pH 5.0 enhanced alignment and filamentous actin stress fiber organization of myoblasts on the surface of scaffolds. The ability to tune topographic features on the surfaces of scaffolds independent of mechanical properties provides a valuable tool for designing microthread-based scaffolds to enhance regeneration of functional muscle tissue.

Skeletal Muscle Tissue Engineering: Biomaterials-Based Strategies for the Treatment of Volumetric Muscle Loss.

Millions of Americans suffer from skeletal muscle injuries annually that can result in volumetric muscle loss (VML), where extensive musculoskeletal damage and tissue loss result in permanent functional deficits. In the case of small-scale injury skeletal muscle is capable of endogenous regeneration through activation of resident satellite cells (SCs). However, this is greatly reduced in VML injuries, which remove native biophysical and biochemical signaling cues and hinder the damaged tissue's ability to direct regeneration. The current clinical treatment for VML is autologous tissue transfer, but graft failure and scar tissue formation leave patients with limited functional recovery. Tissue engineering of instructive biomaterial scaffolds offers a promising approach for treating VML injuries. Herein, we review the strategic engineering of biophysical and biochemical cues in current scaffold designs that aid in restoring function to these preclinical VML injuries. We also discuss the successes and limitations of the three main biomaterial-based strategies to treat VML injuries: acellular scaffolds, cell-delivery scaffolds, and in vitro tissue engineered constructs. Finally, we examine several innovative approaches to enhancing the design of the next generation of engineered scaffolds to improve the functional regeneration of skeletal muscle following VML injuries.

Horseradish Peroxidase-Catalyzed Crosslinking of Fibrin Microthread Scaffolds.

Horseradish peroxidase (HRP) has been investigated as a catalyst to crosslink tissue-engineered hydrogels because of its mild reaction conditions and ability to modulate the mechanical properties of the matrix. Here, we report the results of the first study investigating the use of HRP to crosslink fibrin scaffolds. We examined the effect of varying HRP and hydrogen peroxide (H2O2) incorporation strategies on the resulting crosslink density and structural properties of fibrin in a microthread scaffold format. Primary (1°) and secondary (2°) scaffold modification techniques were evaluated to crosslink fibrin microthread scaffolds. A primary scaffold modification technique was defined as incorporating crosslinking agents into the microthread precursor solutions during extrusion. A secondary scaffold modification technique was defined as incubating the microthreads in a postprocessing crosslinker bath. Fibrin microthreads were enzymatically crosslinked through primary, secondary, or a combination of both approaches. All fibrin microthread scaffolds crosslinked with HRP and H2O2 via primary and/or secondary methods exhibited an increase in dityrosine crosslink density compared with uncrosslinked control microthreads, demonstrated by scaffold fluorescence. Fourier transform infrared spectroscopy indicated the formation of isodityrosine bonds in 1° HRP crosslinked microthreads. Characterization of tensile mechanical properties revealed that all HRP crosslinked microthreads were significantly stronger than control microthreads. Primary (1°) HRP crosslinked microthreads also demonstrated significantly slower degradation than control microthreads, suggesting that incorporating HRP and H2O2 during extrusion yields scaffolds with increased resistance to proteolytic degradation. Finally, cells seeded on HRP crosslinked microthreads retained a high degree of viability, demonstrating that HRP crosslinking yields biocompatible scaffolds that are suitable for tissue engineering. The goal of this work was to facilitate the logical design of enzymatically crosslinked fibrin microthreads with tunable structural properties, enabling their application for engineered tissue constructs with varied mechanical and structural properties.

Designing Biopolymer Microthreads for Tissue Engineering and Regenerative Medicine.

Native tissue structures possess elaborate extracellular matrix (ECM) architectures that inspire the design of fibrous structures in the field of regenerative medicine. We review the literature with respect to the successes and failures, as well as the future promise of biopolymer microthreads as scaffolds to promote endogenous and exogenous tissue regeneration. Biomimetic microthread tissue constructs have been proposed for the functional regeneration of tendon, ligament, skeletal muscle, and ventricular myocardial tissues. To date, biopolymer microthreads have demonstrated promising results as materials to recapitulate the hierarchical structure of simple and complex tissues and well as biochemical signaling cues to direct cell-mediated tissue regeneration. Biopolymer microthreads have also demonstrated exciting potential as a platform technology for the targeted delivery of stem cells and therapeutic molecules. Future studies will focus on the design of microthread-based tissue analogs that strategically integrate growth factors and progenitor cells to temporally direct cell-mediated processes that promote enhanced functional tissue regeneration.