Developing quantitative MRI parameters to characterize host response and tissue ingrowth into collagen scaffolds.

The in vivo evaluation of soft biomaterial implant remodeling routinely requires the surgical removal of the implant for subsequent histological assessment of tissue ingrowth and scaffold remodeling. This approach is very resource intensive, often destructive, and imposes practical limitations on how effectively these materials can be evaluated. MRI has the potential to non-invasively monitor the remodeling of implanted collagen scaffolds in real time. This study investigated the development of a model system to characterize the cellular infiltration, void area fraction, and angiogenesis in collagen scaffold implants using T2 relaxation time and apparent diffusion coefficient (ADC) maps along with conventional histological techniques. Initial correlations found statistically significant relationships between the MRI and histological parameters for various regions of the implanted sponges: T2 versus cell density (r ≈ -0.83); T2 versus void area fraction (r ≈ +0.78); T2 versus blood vessel density (r ≈ +0.95); ADC versus cell density (r ≈ -0.77); and ADC versus void area fraction (r ≈ +0.84). This suggests that MRI is sensitive to specific remodeling parameters and has the potential to serve as a non-invasive tool to monitor the remodeling of implanted collagen scaffolds, and to ultimately assess the ability of these scaffolds to regenerate the functional properties of damaged tissues such as tendons, ligaments, skin or skeletal muscle.

Developing Porous Fibrin Scaffolds with Tunable Anisotropic Features to Direct Myoblast Orientation.

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.

Micropatterned fibrin scaffolds increase cardiomyocyte alignment and contractility for the fabrication of engineered myocardial tissue.

Cardiovascular disease is the leading cause of death in the United States, which can result in blockage of a coronary artery, triggering a myocardial infarction (MI), scar tissue formation in the myocardium, and ultimately heart failure. Currently, the gold-standard solution for total heart failure is a heart transplantation. An alternative to total-organ transplantation is surgically remodeling the ventricle with the implantation of a cardiac patch. Acellular cardiac patches have previously been investigated using synthetic or decellularized native materials to improve cardiac function. However, a limitation of this strategy is that acellular cardiac patches only reshape the ventricle and do not increase cardiac contractile function. Toward the development of a cardiac patch, our laboratory previously developed a cell-populated composite fibrin scaffold and aligned microthreads to recapitulate the mechanical properties of native myocardium. In this study, we explore micropatterning the surfaces of fibrin gels to mimic anisotropic native tissue architecture and promote cellular alignment of human induced pluripotent stem cell cardiomyocytes (hiPS-CM), which is crucial for increasing scaffold contractile properties. hiPS-CMs seeded on micropatterned surfaces exhibit cellular elongation, distinct sarcomere alignment, and circumferential connexin-43 staining at 14 days of culture, which are necessary for mature contractile properties. Constructs were also subject to electrical stimulation during culture to promote increased contractile properties. After 7 days of stimulation, contractile strains of micropatterned constructs were significantly higher than unpatterned controls. These results suggest that the use of micropatterned topographic cues on fibrin scaffolds may be a promising strategy for creating engineered cardiac 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.

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.

Creation of a contractile biomaterial from a decellularized spinach leaf without ECM protein coating: An in vitro study.

Myocardial infarction (MI) results in the death of cardiac tissue, decreases regional contraction, and can lead to heart failure. Tissue engineered cardiac patches containing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) can restore contractile function. However, cells within thick patches require vasculature for blood flow. Recently, we demonstrated fibronectin coated decellularized leaves provide a suitable scaffold for hiPS-CMs. Yet, the necessity of this additional coating step is unclear. Therefore, we compared hiPS-CM behavior on decellularized leaves coated with collagen IV or fibronectin extracellular matrix (ECM) proteins to noncoated leaves for up to 21 days. Successful coating was verified by immunofluorescence. Similar numbers of hiPS-CMs adhered to coated and noncoated decellularized leaves for 21 days. At Day 14, collagen IV coated leaves contracted more than noncoated leaves (3.25 ± 0.39% vs. 1.54 ± 0.60%; p < .05). However, no differences in contraction were found between coated leaves, coated tissue culture plastic (TCP), noncoated leaves, or noncoated TCP at other time points. No significant differences were observed in hiPS-CM spreading or sarcomere lengths on leaves with or without coating. This study demonstrates that cardiac scaffolds can be created from decellularized leaves without ECM coatings. Noncoated decellularized leaf surfaces facilitate robust cell attachment for an engineered tissue patch.

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.

Discrete crosslinked fibrin microthread scaffolds for tissue regeneration.

In this study, we report on the development of discrete fibrin microthreads as well as novel scaffolds composed of arrays of fibrin threads. These scaffolds exhibit mechanical properties that are significantly greater than fibrin gels and cellular responses suggesting that the materials are conducive to the development of organized, aligned tissues. Fibrin microthreads were produced by coextruding solutions of 70 mg/mL fibrinogen and 6 U/mL thrombin through small diameter polyethylene tubing. Uncrosslinked fibrin microthreads averaged 55-65 microm in hydrated diameter and achieved ultimate tensile strengths approaching 4.5 MPa. The strengths and stiffnesses of the microthreads were approximately twofold greater when the materials were treated with exposure to ultraviolet (UV) light. Although UV crosslinking attenuated fibroblast proliferation, uncrosslinked fibrin microthreads supported fibroblast attachment, proliferation, and alignment, suggesting that they represent a viable biomaterial for the aligned regeneration of tissues. Because of the physiologic roles of fibrin matrices in the early phase of wound healing, we anticipate that these fibrin-based microthreads will direct the spatially and temporally complex processes of cell-mediated tissue ingrowth and regeneration.

Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

Crosslinking of discrete self-assembled collagen threads: Effects on mechanical strength and cell-matrix interactions.

Bundles of threads extruded from type I collagen have been researched extensively as scaffolds to promote the repair and regeneration of torn tendons and ligaments. The success of these scaffolds has been limited by insufficient tissue ingrowth from the wound margin, which may be inhibited by the chemical or physical crosslinking treatment used to increase the mechanical properties and decrease the degradation rate of these scaffolds. Recently, self-assembled collagen threads extruded from solutions of type I collagen molecules were shown to possess ultimate tensile strengths and structural properties comparable to native tendon fibers; however the tissue response to these threads has yet to be determined. The goal of this study was to investigate the effects of various crosslinking techniques on the mechanical properties as well as the in vitro rate of new tissue ingrowth on these threads. Our findings indicate that the physical crosslinking techniques, dehydrothermal (DHT) or ultraviolet light (UV), most significantly improve the mechanical strengths of the threads, but most significantly decrease the rate of cell migration. In contrast, carbodiimide (EDC) crosslinking achieved sub-optimal strength generation, but demonstrated improved cell migration rates. Future studies will investigate the design of threads with surface biochemistries that maximize tissue ingrowth while maintaining the mechanical stability of the scaffold.

* Design of an In Vitro Model of Cell Recruitment for Skeletal Muscle Regeneration Using Hepatocyte Growth Factor-Loaded Fibrin Microthreads.

Large skeletal muscle defects that result in volumetric muscle loss (VML) result in the destruction of the basal lamina, which removes key signaling molecules such as hepatocyte growth factor (HGF) from the wound site, eliminating the endogenous capacity of these injuries to regenerate. We recently showed that HGF-loaded fibrin microthreads increased the force production in muscle tissues after 60 days in a mouse VML model. In this study, we created an in vitro, three-dimensional (3D) microscale outgrowth assay system designed to mimic cell recruitment in vivo, and investigated the effect of HGF-loaded, cross-linked fibrin microthreads on myoblast recruitment to predict the results observed in vivo. This outgrowth assay discretely separated the cellular and molecular functions (migration, proliferation, and chemotaxis) that direct outgrowth from the wound margin, creating a powerful platform to model cell recruitment in axially aligned tissues, such as skeletal muscle. The degree of cross-linking was controlled by pH and microthreads cross-linked using physiologically neutral pH (EDCn) facilitated the release of active HGF; increasing the two-dimensional migration and 3D outgrowth of myoblasts twofold. While HGF adsorbed to uncross-linked microthreads, it did not enhance myoblast migration, possibly due to the low concentrations that were adsorbed. Regardless of the amount of HGF adsorbed on the microthreads, myoblast proliferation increased significantly on stiffer, cross-linked microthreads. Together, the results of these studies show that HGF loaded onto EDCn microthreads supported enhanced myoblast migration and recruitment and suggest that our novel outgrowth assay system is a robust in vitro screening tool that predicts the performance of fibrin microthreads in vivo.

Design of a Fibrin Microthread-Based Composite Layer for Use in a Cardiac Patch.

The ability to modulate the mechanical properties, and cell alignment within a cardiac patch without hindering cell functionality may have significant impact on developing therapies for treating myocardial infarctions. We developed fibrin-based composite layers comprising aligned microthreads distributed uniformly throughout a hydrogel. Increasing the microthread volume fraction (∼5%, 11% and 22%) significantly increased the moduli of the scaffolds (20.6 ± 8.1, 46.4 ± 23.0, and 97.5 ± 49.3 kPa, respectively), p < 0.05. Analyses of cell-mediated contractile strains and frequencies showed no significant differences among composite layers and fibrin hydrogel controls, suggesting that microthread-based composite layers exhibit similar active functional properties. Cell orientation in composite layers suggests an increase in nuclear alignment within 100 µm of fibrin microthreads and suggests that microthreads influence the alignment in adjacent areas. In this study, we developed composite layers with tunable, mechanical patch properties that improve cell alignment and support cell functionality.

Delivering stem cells to the healthy heart on biological sutures: effects on regional mechanical function.

Current cardiac cell therapies cannot effectively target and retain cells in a specific area of the heart. Cell-seeded biological sutures were previously developed to overcome this limitation, demonstrating targeted delivery with > 60% cell retention. In this study, both cell-seeded and non-seeded fibrin-based biological sutures were implanted into normal functioning rat hearts to determine the effects on mechanical function and fibrotic response. Human mesenchymal stem cells (hMSCs) were used based on previous work and established cardioprotective effects. Non-seeded or hMSC-seeded sutures were implanted into healthy athymic rat hearts. Before cell seeding, hMSCs were passively loaded with quantum dot nanoparticles. One week after implantation, regional stroke work index and systolic area of contraction (SAC) were evaluated on the epicardial surface above the suture. Cell delivery and retention were confirmed by quantum dot tracking, and the fibrotic tissue area was evaluated. Non-seeded biological sutures decreased SAC near the suture from 0.20 ± 0.01 measured in sham hearts to 0.08 ± 0.02, whereas hMSC-seeded biological sutures dampened the decrease in SAC (0.15 ± 0.02). Non-seeded sutures also displayed a small amount of fibrosis around the sutures (1.0 ± 0.1 mm2 ). Sutures seeded with hMSCs displayed a significant reduction in fibrosis (0.5 ± 0.1 mm2 , p < 0.001), with quantum dot-labelled hMSCs found along the suture track. These results show that the addition of hMSCs attenuates the fibrotic response observed with non-seeded sutures, leading to improved regional mechanics of the implantation region. Copyright © 2014 John Wiley & Sons, Ltd.

Multiphoton excited fabricated nano and micro patterned extracellular matrix proteins direct cellular morphology.

We use multiphoton excited (MPE) photochemistry to fabricate patterned extracellular matrices (ECM) and to investigate the morphology of human dermal fibroblasts adhered to the resulting photocrosslinked linear structures of fibronectin (FN), fibrinogen (FG), and bovine serum albumin (BSA). These proteins were chosen to systematically investigate the roles of topography and ECM biochemistry on cell spreading, as fibroblasts bind directly to both FN and FG at RGD sites through known integrins, whereas BSA provides no comparable ECM cues for cell binding. MPE crosslinked patterns are created from parallel linear structures 600 nm in width, 200 microm in length, and spaced by either 10 or 40 microm. Immunofluorescence staining of FN and FG was used to assay the functionality of crosslinked proteins. The metrics of orientation, elongation, and cell perimeter were used to quantitate the resulting cellular behavior on the crosslinked protein patterns. These parameters all reflect statistical differences for cells on BSA, relative to the similar statistical behavior on fibronectin and fibrinogen. Cells on the BSA patterns are constrained by physical guidance and orientation between linear structures. In contrast, cells adhered on both FN and FG had a greater propensity to spread across adjacent structures, indicating the importance of cell matrix interactions. Focal adhesion staining of cells adhered to the protein structures revealed similar trends. These findings are consistent with our hypothesis that these crosslinked matrix protein structures are expected to direct cell adhesion and spreading and that the topography and ECM cues lead to different forms of guidance.

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.

The Effect of Sterilization Methods on the Structural and Chemical Properties of Fibrin Microthread Scaffolds.

A challenge for the design of scaffolds in tissue engineering is to determine a terminal sterilization method that will retain the structural and biochemical properties of the materials. Since commonly used heat and ionizing energy-based sterilization methods have been shown to alter the material properties of protein-based scaffolds, the effects of ethanol and ethylene oxide (EtO) sterilization on the cellular compatibility and the structural, chemical, and mechanical properties of uncrosslinked, UV crosslinked, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) crosslinked fibrin microthreads in neutral (EDCn) or acidic (EDCa) buffers are evaluated. EtO sterilization significantly reduces the tensile strength of uncrosslinked microthreads. Surface chemistry analyses show that EtO sterilization induces alkylation of EDCa microthreads leading to a significant reduction in myoblast attachment. The material properties of EDCn microthreads do not appear to be affected by the sterilization method. These results significantly enhance the understanding of how sterilization or crosslinking techniques affect the material properties of protein scaffolds.

Novel Conductive Carbon Black and Polydimethlysiloxane ECG Electrode: A Comparison with Commercial Electrodes in Fresh, Chlorinated, and Salt Water.

In this study, we evaluated the performance of two novel conductive carbon black (CB) and polydimethlysiloxane (PDMS) bio-potential electrodes, with and without an integrated flexible copper mesh, against commercially available electrodes (Polar(®) textile, Silver-coated textile, and carbon rubber). The electrodes were tested in three types of water (fresh/unfiltered, chlorinated, and salt water). Our testing revealed that our CB/PDMS electrode with integrated copper mesh provided a high-fidelity ECG signal morphologies without any amplitude degradation in all of the types of water tested (N = 10). The non-meshed CB/PDMS electrodes were also subjected to a long-term durability test by the US Navy SCUBA divers during which the electrodes maintained ECG signal quality for a 6 h period of continuous use. The results of a material degradation analysis revealed the CB/PDMS composite material does not exhibit significant changes in physical integrity after prolonged exposure to the test conditions. The newly developed meshed CB/PDMS electrodes have the potential to be used in a wide variety of both dry and wet environments including the challenge of obtaining ECG signals in salt water environments.

The influence of microtextured basal lamina analog topography on keratinocyte function and epidermal organization.

The rational design of future bioengineered skin substitutes requires an understanding of the mechanisms by which the three-dimensional microarchitecture of tissue scaffolds modulates keratinocyte function. Microtextured basal lamina analogs were developed to investigate the relationship between the characteristic topography at the dermal-epidermal interface of native skin and keratinocyte function. Microfabrication techniques were used to create master patterns, negative replicates, and collagen membranes with ridges and channels of length scales (e.g., grooves of 50-200 microm in depth and width) similar to the invaginations found in basal lamina at the dermal-epidermal junction of native skin. Keratinocytes were seeded on the surfaces of basal lamina analogs, and histological analyses were performed after 7 days of tissue culture at the air-liquid interface. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microtextured membranes. Morphometric analyses of immunostained skin equivalents suggest that keratinocyte stratification and differentiation increases as channel depth increases and channel width decreases. This trend was most pronounced in channels with the highest depth-to-width ratios (i.e., 200 microm deep, 50 microm wide). It is anticipated that the findings from these studies will elucidate design parameters to enhance the performance of future bioengineered skin substitutes.

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

Skeletal muscle injuries typically result from traumatic incidents such as combat injuries where soft-tissue extremity injuries are present in one of four cases. Further, about 4.5 million reconstructive surgical procedures are performed annually as a result of car accidents, cancer ablation, or cosmetic procedures. These combat- and trauma-induced skeletal muscle injuries are characterized by volumetric muscle loss (VML), which significantly reduces the functionality of the injured muscle. While skeletal muscle has an innate repair mechanism, it is unable to compensate for VML injuries because large amounts of tissue including connective tissue and basement membrane are removed or destroyed. This results in a significant need to develop off-the-shelf biomimetic scaffolds to direct skeletal muscle regeneration. Here, the structure and organization of native skeletal muscle tissue is described in order to reveal clear design parameters that are necessary for scaffolds to mimic in order to successfully regenerate muscular tissue. We review the literature with respect to the materials and methodologies used to develop scaffolds for skeletal muscle tissue regeneration as well as the limitations of these materials. We further discuss the variety of cell sources and different injury models to provide some context for the multiple approaches used to evaluate these scaffold materials. Recent findings are highlighted to address the state of the field and directions are outlined for future strategies, both in scaffold design and in the use of different injury models to evaluate these materials, for regenerating functional skeletal muscle. STATEMENT OF SIGNIFICANCE: Volumetric muscle loss (VML) injuries result from traumatic incidents such as those presented from combat missions, where soft-tissue extremity injuries are represented in one of four cases. These injuries remove or destroy large amounts of skeletal muscle including the basement membrane and connective tissue, removing the structural, mechanical, and biochemical cues that usually direct its repair. This results in a significant need to develop off-the-shelf biomimetic scaffolds to direct skeletal muscle regeneration. In this review, we examine current strategies for the development of scaffold materials designed for skeletal muscle regeneration, highlighting advances and limitations associated with these methodologies. Finally, we identify future approaches to enhance skeletal muscle regeneration.

Rapid release of growth factors regenerates force output in volumetric muscle loss injuries.

A significant challenge in the design and development of biomaterial scaffolds is to incorporate mechanical and biochemical cues to direct organized tissue growth. In this study, we investigated the effect of hepatocyte growth factor (HGF) loaded, crosslinked fibrin (EDCn-HGF) microthread scaffolds on skeletal muscle regeneration in a mouse model of volumetric muscle loss (VML). The rapid, sustained release of HGF significantly enhanced the force production of muscle tissue 60 days after injury, recovering more than 200% of the force output relative to measurements recorded immediately after injury. HGF delivery increased the number of differentiating myoblasts 14 days after injury, and supported an enhanced angiogenic response. The architectural morphology of microthread scaffolds supported the ingrowth of nascent myofibers into the wound site, in contrast to fibrin gel implants which did not support functional regeneration. Together, these data suggest that EDCn-HGF microthreads recapitulate several of the regenerative cues lost in VML injuries, promote remodeling of functional muscle tissue, and enhance the functional regeneration of skeletal muscle. Further, by strategically incorporating specific biochemical factors and precisely tuning the structural and mechanical properties of fibrin microthreads, we have developed a powerful platform technology that may enhance regeneration in other axially aligned tissues.