Aligned Collagen Sponges with Tunable Pore Size for Skeletal Muscle Tissue Regeneration.

Volumetric muscle loss (VML) is a traumatic injury where at least 20% of the mass of a skeletal muscle has been destroyed and functionality is lost. The standard treatment for VML, autologous tissue transfer, is limited as approximately 1 in 10 grafts fail because of necrosis or infection. Tissue engineering strategies seek to develop scaffolds that can regenerate injured muscles and restore functionality. Many of these scaffolds, however, are limited in their ability to restore muscle functionality because of an inability to promote the alignment of regenerating myofibers. For aligned myofibers to form on a scaffold, myoblasts infiltrate the scaffold and receive topographical cues to direct targeted myofiber growth. We seek to determine the optimal pore size for myoblast infiltration and differentiation. We developed a method of tuning the pore size within collagen scaffolds while inducing longitudinal alignment of these pores. Significantly different pore sizes were generated by adjusting the freezing rate of the scaffolds. Scaffolds frozen at -20 °C contained the largest pores. These scaffolds promoted the greatest level of cell infiltration and orientation in the direction of pore alignment. Further research will be conducted to induce higher levels of myofiber formation, to ultimately create an off-the-shelf treatment for VML injuries.

Porous biomaterial scaffolds for skeletal muscle tissue engineering.

Volumetric muscle loss is a traumatic injury which overwhelms the innate repair mechanisms of skeletal muscle and results in significant loss of muscle functionality. Tissue engineering seeks to regenerate these injuries through implantation of biomaterial scaffolds to encourage endogenous tissue formation and to restore mechanical function. Many types of scaffolds are currently being researched for this purpose. Scaffolds are typically made from either natural, synthetic, or conductive polymers, or any combination therein. A major criterion for the use of scaffolds for skeletal muscle is their porosity, which is essential for myoblast infiltration and myofiber ingrowth. In this review, we summarize the various methods of fabricating porous biomaterial scaffolds for skeletal muscle regeneration, as well as the various types of materials used to make these scaffolds. We provide guidelines for the fabrication of scaffolds based on functional requirements of skeletal muscle tissue, and discuss the general state of the field for skeletal muscle tissue engineering.

Jointly Optimized Spatial Histogram UNET Architecture (JOSHUA) for Adipose Tissue Segmentation.

Objective. We aim to develop a machine learning algorithm to quantify adipose tissue deposition at surgical sites as a function of biomaterial implantation. Impact Statement. To our knowledge, this study is the first investigation to apply convolutional neural network (CNN) models to identify and segment adipose tissue in histological images from silk fibroin biomaterial implants. Introduction. When designing biomaterials for the treatment of various soft tissue injuries and diseases, one must consider the extent of adipose tissue deposition. In this work, we analyzed adipose tissue accumulation in histological images of sectioned silk fibroin-based biomaterials excised from rodents following subcutaneous implantation for 1, 2, 4, or 8 weeks. Current strategies for quantifying adipose tissue after biomaterial implantation are often tedious and prone to human bias during analysis. Methods. We used CNN models with novel spatial histogram layer(s) that can more accurately identify and segment regions of adipose tissue in hematoxylin and eosin (H&E) and Masson's trichrome stained images, allowing for determination of the optimal biomaterial formulation. We compared the method, Jointly Optimized Spatial Histogram UNET Architecture (JOSHUA), to the baseline UNET model and an extension of the baseline model, attention UNET, as well as to versions of the models with a supplemental attention-inspired mechanism (JOSHUA+ and UNET+). Results. The inclusion of histogram layer(s) in our models shows improved performance through qualitative and quantitative evaluation. Conclusion. Our results demonstrate that the proposed methods, JOSHUA and JOSHUA+, are highly beneficial for adipose tissue identification and localization. The new histological dataset and code used in our experiments are publicly available.

Self-assembling Peptide Hydrogels Facilitate Vascularization in Two-Component Scaffolds.

One of the major constraints against using polymeric scaffolds as tissue-regenerative matrices is a lack of adequate implant vascularization. Self-assembling peptide hydrogels can sequester small molecules and biological macromolecules, and they can support infiltrating cells in vivo. Here we demonstrate the ability of self-assembling peptide hydrogels to facilitate angiogenic sprouting into polymeric scaffolds after subcutaneous implantation. We constructed two-component scaffolds that incorporated microporous polymeric scaffolds and viscoelastic nanoporous peptide hydrogels. Nanofibrous hydrogels modified the biocompatibility and vascular integration of polymeric scaffolds with microscopic pores (pore diameters: 100-250 µm). In spite of similar amphiphilic sequences, charges, secondary structures, and supramolecular nanostructures, two soft hydrogels studied herein had different abilities to aid implant vascularization, but had similar levels of cellular infiltration. The functional difference of the peptide hydrogels was predicted by the difference in the bioactive moieties inserted into the primary sequences of the peptide monomers. Our study highlights the utility of soft supramolecular hydrogels to facilitate host-implant integration and control implant vascularization in biodegradable polyester scaffolds in vivo. Our study provides useful tools in designing multi-component regenerative scaffolds that recapitulate vascularized architectures of native tissues.

Recent Trends in Injury Models to Study Skeletal Muscle Regeneration and Repair.

Skeletal muscle injuries that occur from traumatic incidents, such as those caused by car accidents or surgical resections, or from injuries sustained on the battlefield, result in the loss of functionality of the injured muscle. To understand skeletal muscle regeneration and to better treat these large scale injuries, termed volumetric muscle loss (VML), in vivo injury models exploring the innate mechanisms of muscle injury and repair are essential for the creation of clinically applicable treatments. While the end result of a muscle injury is often the destruction of muscle tissue, the manner in which these injuries are induced as well as the response from the innate repair mechanisms found in muscle in each animal models can vary. This targeted review describes injury models that assess both skeletal muscle regeneration (i.e., the response of muscle to myotoxin or ischemic injury) and skeletal muscle repair (i.e., VML injury). We aimed to summarize the injury models used in the field of skeletal muscle tissue engineering, paying particular attention to strategies to induce muscle damage and how to standardize injury conditions for future experiments.

Hyperosmolar potassium inhibits myofibroblast conversion and reduces scar tissue formation.

Scar formation is a natural result of almost all wound healing in adult mammals. Unfortunately, scarring disrupts normal tissue function and can cause significant physical and psychological distress. In addition to improving surgical techniques to limit scar formation, several therapies are under development towards the same goal. Many of these treatments aim to disrupt transforming growth factor ß1 (TGFß1) signaling, as this is a critical control point for fibroblast differentiation into myofibroblasts; a contractile cell that organizes synthesized collagen fibrils into scar tissue. The present study aimed to examine the role of hyperosmolar potassium gluconate (KGluc) on fibroblast function in skin repair. KGluc was first determined to negatively regulate fibroblast proliferation, metabolism, and migration in a dose-dependent manner in vitro. Increasing concentrations of KGluc also inhibited differentiation into myofibroblasts, suggesting that local KGluc treatment might reduce fibrosis. KGluc delivery was confirmed via loading into collagen hydrogels and used to treat a full thickness skin wound in mice. KGluc qualitatively slowed initial closure of the wounds and resulted in tissue that more closely resembled mature, healthy skin (epidermal thickness and dermal-epidermal morphology) when compared to unloaded collagen hydrogels. KGluc treatment significantly reduced the number of myofibroblasts within the dermis while upregulated blood vessel density with respect to unloaded hydrogels, likely a result of disruption of TGFß1 signaling. Taken together, these data demonstrate the effectiveness of KGluc treatment on skin wound healing and suggest that this may be an efficient treatment to limit scar formation.

Tissue Models for Neurogenesis and Repair in 3D.

Development and maturation of vascular and neuronal tissues occurs simultaneously in utero, and are regulated by significant crosstalk. We report on the development of a 3D tissue system to model neurogenesis and recapitulate developmental signaling conditions. Human umbilical vein endothelial cells (HUVECs) were seeded inside channels within collagen gels to represent nascent vascular networks. Axons extending from chicken dorsal root ganglia (DRGs) grew significantly longer and preferentially towards the HUVEC seeded channels with respect to unloaded channels. To replicate these findings without the vascular component, channels were loaded with brain-derived neurotrophic factor (BDNF), the principle signaling molecule in HUVEC-stimulated axonal growth, and axons likewise were significantly longer and grew preferentially towards the BDNF-loaded channels with respect to controls. This 3D tissue system was then used as an in vitro replicate for peripheral nerve injury, with neural repair observed within 2 weeks. These results demonstrate that our 3D tissue system can model neural network formation, repair after laceration injuries, and can be utilized to further study how these networks form and interact with other tissues, such as skin or skeletal muscle.

Human endothelial cells secrete neurotropic factors to direct axonal growth of peripheral nerves.

Understanding how nerves spontaneously innervate tissues or regenerate small injuries is critical to enhance material-based interventions to regenerate large scale, traumatic injuries. During embryogenesis, neural and vascular tissues form interconnected, complex networks as a result of signaling between these tissue types. Here, we report that human endothelial cells (HUVECs) secrete brain-derived neurotrophic factor (BDNF), which significantly stimulated axonal growth from chicken or rat dorsal root ganglia (DRGs). HUVEC-conditioned medium was sufficient to enhance axonal growth, demonstrating that direct cell-cell contact was not required. When BDNF was neutralized, there was a significant reduction in axonal growth when incubated in HUVEC-conditioned medium and in direct co-culture with HUVECs. These data show that HUVECs secrete neurotrophic factors that significantly enhance axonal growth, and can inform future in vivo studies to direct or pattern the angiogenic response in regenerating tissues to encourage re-innervation.

* 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.

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.

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.

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.

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

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 3-fold, 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 2-fold 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.

Crosslinking strategies facilitate tunable structural properties of fibrin microthreads.

A significant challenge in the design of biomimetic scaffolds is combining morphologic, mechanical, and biochemical cues into a single construct to promote tissue regeneration. In this study, we analyzed the effects of different crosslinking conditions on fibrin biopolymer microthreads to create morphologic scaffolds with tunable mechanical properties that are designed for directional cell guidance. Fibrin microthreads were crosslinked using carbodiimides in either acidic or neutral buffer, and the mechanical, structural, and biochemical responses of the microthreads were investigated. Crosslinking in the presence of acidic buffer (EDCa) created microthreads that had significantly higher tensile strengths and moduli than all other microthreads, and failed at lower strains than all other microthreads. Microthreads crosslinked in neutral buffer (EDCn) were also significantly stronger and stiffer than uncrosslinked threads and were comparable to contracting muscle in stiffness. Swelling ratios of crosslinked microthreads were significantly different from each other and uncrosslinked controls, suggesting a difference in the internal organization and compaction of the microthreads. Using an in vitro degradation assay, we observed that EDCn microthreads degraded within 24h, six times slower than uncrosslinked control threads, but EDCa microthreads did not show any significant indication of degradation within the 7-day assay period. Microthreads with higher stiffnesses supported significantly increased attachment of C2C12 cells, as well as increases in cell proliferation without a decrease in cell viability. Taken together, these data demonstrate the ability to create microthreads with tunable mechanical and structural properties that differentially direct cellular functions. Ultimately, we anticipate that we can strategically exploit these properties to promote site-specific tissue regeneration.

Oxygen diffusivity of biologic and synthetic scaffold materials for tissue engineering.

Scaffolds for tissue engineering and regenerative medicine applications are commonly manufactured from synthetic materials, intact or isolated components of extracellular matrix (ECM), or a combination of such materials. After surgical implantation, the metabolic requirements of cells that populate the scaffold depend upon adequate gas and nutrient exchange with the surrounding microenvironment. The present study measured the oxygen transfer through three biologic scaffold materials composed of ECM including small intestinal submucosa (SIS), urinary bladder submucosa (UBS), and urinary bladder matrix (UBM), and one synthetic biomaterial, Dacron. The oxygen diffusivity was calculated from Fick's first law of diffusion. Each material permitted measurable oxygen diffusion. The diffusivity of SIS was found to be dependent on the direction of oxygen transfer; the oxygen transfer in the abluminal-to-luminal direction was significantly greater than the luminal-to-abluminal direction. The oxygen diffusivity of UBM and UBS were similar despite the presence of an intact basement membrane on the luminal surface of UBM. Dacron showed oxygen diffusivity values seven times greater than the ECM biomaterials. The current study showed that each material has unique oxygen diffusivity values, and these values may be dependent on the scaffold's ultrastructure.