Engineering skeletal muscle: Building complexity to achieve functionality.

Volumetric muscle loss (VML) VML is defined as the loss of a critical mass of skeletal muscle that overwhelms the muscle's natural healing mechanisms, leaving patients with permanent functional deficits and deformity. The treatment of these defects is complex, as skeletal muscle is a composite structure that relies closely on the action of supporting tissues such as tendons, vasculature, nerves, and bone. The gold standard of treatment for VML injuries, an autologous muscle flap transfer, suffers from many shortcomings but nevertheless remains the best clinically available avenue to restore function. This review will consider the use of composite tissue engineered constructs, with multiple components that act together to replicate the function of an intact muscle, as an alternative to autologous muscle flaps. We will discuss recent advances in the field of tissue engineering that enable skeletal muscle constructs to more closely reproduce the functionality of an autologous muscle flap by incorporating vasculature, promoting innervation, and reconstructing the muscle-tendon boundary. Additionally, our understanding of the cellular composition of skeletal muscle has evolved to recognize the importance of a diverse variety of cell types in muscle regeneration, including fibro/adipogenic progenitors and immune cells like macrophages and regulatory T cells. We will address recent advances in our understanding of how these cell types interact with, and can be incorporated into, implanted tissue engineered constructs.

Adipogenic-Myogenic Signaling in Engineered Human Muscle Grafts used to Treat Volumetric Muscle Loss.

Tissue-engineered muscle grafts (TEMGs) are a promising treatment for volumetric muscle loss (VML). In this study, human myogenic progenitors (hMPs) cultured on electrospun fibrin microfiber bundles and evaluated the therapeutic potential of engineered hMP TEMGs in the treatment of murine tibialis anterior (TA) VML injuries is employed. In vitro, the hMP TEMGs express mature muscle markers by 21 days. Upon implantation into VML injuries, the hMP TEMGs enable remarkable regeneration. To further promote wound healing and myogenesis, human adipose-derived stem/stromal cells (hASCs) as fibroadipogenic progenitor (FAP)-like cells with the potential to secrete pro-regenerative cytokines are incorporated. The impact of dose and timing of seeding the hASCs on in vitro myogenesis and VML recovery using hMP-hASC TEMGs are investigated. The hASCs increase myogenesis of hMPs when co-cultured at 5% hASCs: 95% hMPs and with delayed seeding. Upon implantation into immunocompromised mice, hMP-hASC TEMGs increase cell survival, collagen IV deposition, and pro-regenerative macrophage recruitment, but result in excessive adipose tissue growth after 28 days. These data demonstrate the interactions of hASCs and hMPs enhance myogenesis in vitro but there remains a need to optimize treatments to minimize adipogenesis and promote full therapeutic recovery following VML treatment.

Point-of-care treatment of geometrically complex midfacial critical-sized bone defects with 3D-Printed scaffolds and autologous stromal vascular fraction.

Critical-sized midfacial bone defects present a unique clinical challenge due to their complex three-dimensional shapes and intimate associations with sensory organs. To address this challenge, a point-of-care treatment strategy for functional, long-term regeneration of 2 cm full-thickness segmental defects in the zygomatic arches of Yucatan minipigs is evaluated. A digital workflow is used to 3D-print anatomically precise, porous, biodegradable scaffolds from clinical-grade poly-ε-caprolactone and decellularized bone composites. The autologous stromal vascular fraction of cells (SVF) is isolated from adipose tissue extracts and infused into the scaffolds that are implanted into the zygomatic ostectomies. Bone regeneration is assessed up to 52 weeks post-operatively in acellular (AC) and SVF groups (BV/DV = 0.64 ± 0.10 and 0.65 ± 0.10 respectively). In both treated groups, bone grows from the adjacent tissues and restores the native anatomy. Significantly higher torque is required to fracture the bone-scaffold interface in the SVF (7.11 ± 2.31 N m) compared to AC groups (2.83 ± 0.23 N m). Three-dimensional microcomputed tomography analysis reveals two distinct regenerative patterns: osteoconduction along the periphery of scaffolds to form dense lamellar bone and small islands of woven bone deposits growing along the struts in the scaffold interior. Overall, this study validates the efficacy of using 3D-printed bioactive scaffolds with autologous SVF to restore geometrically complex midfacial bone defects of clinically relevant sizes while also highlighting remaining challenges to be addressed prior to clinical translation.

Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice.

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.

Recent advances toward understanding the role of transplanted stem cells in tissue-engineered regeneration of musculoskeletal tissues.

Stem cell-based tissue engineering is poised to revolutionize the treatment of musculoskeletal injuries. However, in order to overcome scientific, practical, and regulatory obstacles and optimize therapeutic strategies, it is essential to better understand the mechanisms underlying the pro-regenerative effects of stem cells. There has been an attempted paradigm shift within the last decade to think of transplanted stem cells as "medicinal" therapies that orchestrate healing on the basis of their secretome and immunomodulatory profiles rather than acting as bona fide stem cells that proliferate, differentiate, and directly produce matrix to form de novo tissues. Yet the majority of current bone and skeletal muscle tissue engineering strategies are still premised on a direct contribution of stem cells as building blocks to tissue regeneration. Our review of the recent literature finds that researchers continue to focus on the quantification of de novo bone/skeletal muscle tissue following treatment and few studies aim to address this mechanistic conundrum directly. The dichotomy of thought is reflected in the diversity of new advances ranging from in situ three-dimensional bioprinting to a focus on exosomes and extracellular vesicles. However, recent findings elucidating the role of the immune system in tissue regeneration combined with novel imaging platform technologies will have a profound impact on our future understanding of how stem cells promote healing following biomaterial-mediated delivery to defect sites.

Biomonitoring of Polycyclic Aromatic Hydrocarbon Deposition in Greenland Using Historical Moss Herbarium Specimens Shows a Decrease in Pollution During the 20th Century.

Although most point sources of persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs), are at lower latitudes, the Arctic region is contaminated. In particular, PAHs now dominate the POP body burden of the region's marine biota at the lower trophic levels. Greenlandic Inuits have the most elevated levels of POPs in their blood compared to any other population, due to their consumption of seal meat and other marine mammals. PAHs, the by-products of the incomplete combustion of petroleum products, are known carcinogens and have been shown to affect the immune system, reproduction, endocrine functions, and the nervous system. With industrial activities and climate change set to increase local PAH emissions, it is paramount to document changes in atmospheric PAH deposition to further investigate PAH exposure in the region and attribute contaminations to their sources. As a measure of atmospheric pollution, we sampled bryophyte herbarium specimens of three common and widespread species collected in Greenland between the 1920s and 1970s after which time new collections were not available. They were analyzed for 19 PAHs using GC-MS (gas chromatography mass spectrometry). The presence of more low-molecular-weight PAHs than high-molecular-weight PAHs is evidence that the PAH contamination in Greenland is due to long-range transport rather than originating from local sources. The results show peaks in PAH atmospheric deposition in the first part of the 19th century followed by a trend of decrease, which mirror global trends in atmospheric pollution known from those periods. PAHs associated with wood and fossil-fuel combustion decrease in the 1970s coinciding with the disappearance of charcoal pits and foundries in Europe and North America, and a shift away from domestic heating with wood during the 19th century. The results highlight the value of bryophytes as bioindicators to measure PAH atmospheric pollution as well as the unrealized potential of herbaria as historical records of environmental change.

Blood-brain barrier dysfunction in aging induces hyperactivation of TGFß signaling and chronic yet reversible neural dysfunction.

Aging involves a decline in neural function that contributes to cognitive impairment and disease. However, the mechanisms underlying the transition from a young-and-healthy to aged-and-dysfunctional brain are not well understood. Here, we report breakdown of the vascular blood-brain barrier (BBB) in aging humans and rodents, which begins as early as middle age and progresses to the end of the life span. Gain-of-function and loss-of-function manipulations show that this BBB dysfunction triggers hyperactivation of transforming growth factor-ß (TGFß) signaling in astrocytes, which is necessary and sufficient to cause neural dysfunction and age-related pathology in rodents. Specifically, infusion of the serum protein albumin into the young rodent brain (mimicking BBB leakiness) induced astrocytic TGFß signaling and an aged brain phenotype including aberrant electrocorticographic activity, vulnerability to seizures, and cognitive impairment. Furthermore, conditional genetic knockdown of astrocytic TGFß receptors or pharmacological inhibition of TGFß signaling reversed these symptomatic outcomes in aged mice. Last, we found that this same signaling pathway is activated in aging human subjects with BBB dysfunction. Our study identifies dysfunction in the neurovascular unit as one of the earliest triggers of neurological aging and demonstrates that the aging brain may retain considerable latent capacity, which can be revitalized by therapeutic inhibition of TGFß signaling.