A Prospective Evaluation of Infant Cerebellar-Cerebral Functional Connectivity in Relation to Behavioral Development in Autism Spectrum Disorder.

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder diagnosed based on social impairment, restricted interests, and repetitive behaviors. Contemporary theories posit that cerebellar pathology contributes causally to ASD by disrupting error-based learning (EBL) during infancy. The present study represents the first test of this theory in a prospective infant sample, with potential implications for ASD detection. Methods: Data from the Infant Brain Imaging Study (n = 94, 68 male) were used to examine 6-month cerebellar functional connectivity magnetic resonance imaging in relation to later (12/24-month) ASD-associated behaviors and outcomes. Hypothesis-driven univariate analyses and machine learning-based predictive tests examined cerebellar-frontoparietal network (FPN; subserves error signaling in support of EBL) and cerebellar-default mode network (DMN; broadly implicated in ASD) connections. Cerebellar-FPN functional connectivity was used as a proxy for EBL, and cerebellar-DMN functional connectivity provided a comparative foil. Data-driven functional connectivity magnetic resonance imaging enrichment examined brain-wide behavioral associations, with post hoc tests of cerebellar connections. Results: Cerebellar-FPN and cerebellar-DMN connections did not demonstrate associations with ASD. Functional connectivity magnetic resonance imaging enrichment identified 6-month correlates of later ASD-associated behaviors in networks of a priori interest (FPN, DMN), as well as in cingulo-opercular (also implicated in error signaling) and medial visual networks. Post hoc tests did not suggest a role for cerebellar connections. Conclusions: We failed to identify cerebellar functional connectivity-based contributions to ASD. However, we observed prospective correlates of ASD-associated behaviors in networks that support EBL. Future studies may replicate and extend network-level positive results, and tests of the cerebellum may investigate brain-behavior associations at different developmental stages and/or using different neuroimaging modalities.

Infant Visual Brain Development and Inherited Genetic Liability in Autism.

OBJECTIVE: Autism spectrum disorder (ASD) is heritable, and younger siblings of ASD probands are at higher likelihood of developing ASD themselves. Prospective MRI studies of siblings report that atypical brain development precedes ASD diagnosis, although the link between brain maturation and genetic factors is unclear. Given that familial recurrence of ASD is predicted by higher levels of ASD traits in the proband, the authors investigated associations between proband ASD traits and brain development among younger siblings. METHODS: In a sample of 384 proband-sibling pairs (89 pairs concordant for ASD), the authors examined associations between proband ASD traits and sibling brain development at 6, 12, and 24 months in key MRI phenotypes: total cerebral volume, cortical surface area, extra-axial cerebrospinal fluid, occipital cortical surface area, and splenium white matter microstructure. Results from primary analyses led the authors to implement a data-driven approach using functional connectivity MRI at 6 months. RESULTS: Greater levels of proband ASD traits were associated with larger total cerebral volume and surface area and larger surface area and reduced white matter integrity in components of the visual system in siblings who developed ASD. This aligned with weaker functional connectivity between several networks and the visual system among all siblings during infancy. CONCLUSIONS: The findings provide evidence that specific early brain MRI phenotypes of ASD reflect quantitative variation in familial ASD traits. Multimodal anatomical and functional convergence on cortical regions, fiber pathways, and functional networks involved in visual processing suggest that inherited liability has a role in shaping the prodromal development of visual circuitry in ASD.

Aligned nanofibers of decellularized muscle extracellular matrix for volumetric muscle loss.

Volumetric muscle loss (VML) is a traumatic loss of muscle tissue that results in chronic functional impairment. When injured, skeletal muscle is capable of small-scale repair; however, regenerative capacities are lost with VML due to a critical loss stem cells and extracellular matrix (ECM). Consequences of VML include either long-term disability or delayed amputations of the affected limb. While the prevalence of VML is substantial, currently a successful clinical therapy has not been identified. In a previous study, an electrospun composed of polycaprolactone (PCL) and decellularized-ECM (D-ECM) supported satellite cell-mediated myogenic activity in vitro. In this study, we investigate the extent to which this electrospun scaffold can support functional muscle regeneration in a murine model of VML. Experimental groups included no treatment, pure PCL treated, and PCL:D-ECM (50:50 blend) treated VML defects. The PCL:D-ECM scaffold treated VML muscles supported increased activity of anti-inflammatory M2 macrophages (arginase+ ) at Day 28, compared to other experimental groups. Increased myofiber (MHC+ ) regeneration was observed histologically at both Days 7 and 28 post-trauma in blend scaffold treated group compared to PCL treated and untreated groups. However, improvements in muscle weights and force production were not observed. Future studies would evaluate muscle function at longer time-points post-VML injury to allow sufficient time for reinnervation of regenerated muscle fibers.

Regulation of Myogenic Activity by Substrate and Electrical Stimulation In Vitro.

Skeletal muscle has a remarkable regenerative capacity in response to mild injury. However, when muscle is severely injured, muscle regeneration is impaired due to the loss of muscle-resident stem cells, known as satellite cells. Fibrotic tissue, primarily comprising collagen I (COL), is deposited with this critical loss of muscle. In recent studies, supplementation of laminin (LM)-111 has been shown to improve skeletal muscle regeneration in several models of disease and injury. Additionally, electrical stimulation (E-stim) has been investigated as a possible rehabilitation therapy to improve muscle's functional recovery. This study investigated the role of E-stim and substrate in regulating myogenic response. C2C12 myoblasts were allowed to differentiate into myotubes on COL- and LM-coated polydimethylsiloxane molds. The myotubes were subjected to E-stim and compared with nonstimulated controls. While E-stim resulted in increased myogenic activity, irrespective of substrate, LM supported increased proliferation and uniform distribution of C2C12 myoblasts. In addition, C2C12 myoblasts cultured on LM showed higher Sirtuin 1, mammalian target of rapamycin, desmin, nitric oxide, and vascular endothelial growth factor expression. Taken together, these results suggest that an LM substrate is more conducive to myoblast growth and differentiation in response to E-stim in vitro.

Decellularized extracellular matrix gelloids support mesenchymal stem cell growth and function in vitro.

Volumetric muscle loss (VML) injuries are irrecoverable due to a significant loss of regenerative elements, persistent inflammation, extensive fibrosis, and functional impairment. When used in isolation, previous stem cell and biomaterial-based therapies have failed to regenerate skeletal muscle at clinically relevant levels. The extracellular matrix (ECM) microenvironment is crucial for the viability, stemness, and differentiation of stem cells. Decellularized-ECM (D-ECM) scaffolds are at the forefront of ongoing research to develop a viable therapy for VML. Due to the retention of key ECM components, D-ECM scaffolds provide an excellent substrate for the adhesion and migration of several cell types. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and are currently under investigation in clinical trials for a wide range of medical conditions. However, a major limitation to the use of MSCs in clinical applications is their poor viability at the site of transplantation. In this study, we have fabricated spherical scaffolds composed of gelatin and skeletal muscle D-ECM for the adhesion and delivery of MSCs to the site of VML injury. These spherical scaffolds termed "gelloids" supported MSC survival, expansion, trophic factor secretion, immunomodulation, and myogenic protein expression in vitro. Future studies would determine the therapeutic efficacy of this approach in a murine model of VML injury.

Aligned nanofibers of decellularized muscle ECM support myogenic activity in primary satellite cells in vitro.

Volumetric muscle loss (VML) is a loss of over ∼10% of muscle mass that results in functional impairment. Although skeletal muscle possesses the ability to repair and regenerate itself following minor injuries, VML injuries are irrecoverable. Currently, there are no successful clinical therapies for the treatment of VML. Previous studies have treated VML defects with decellularized extracellular matrix (D-ECM) scaffolds derived from either pig urinary bladder or small intestinal submucosa. These therapies were unsuccessful due to the poor mechanical stability of D-ECM leading to quick degradation in vivo. To circumvent these issues, in this manuscript aligned nanofibers of D-ECM were created using electrospinning that mimicked native muscle architecture and provided topographical cues to primary satellite cells. Additionally, combining D-ECM with polycaprolactone (PCL) improved the tensile mechanical properties of the electrospun scaffold. In vitro testing shows that the electrospun scaffold with aligned nanofibers of PCL and D-ECM supports satellite cell growth, myogenic protein expression, and myokine production.

Biomaterial and stem cell-based strategies for skeletal muscle regeneration.

Adult skeletal muscle can regenerate effectively after mild physical or chemical insult. Muscle trauma or disease can overwhelm this innate capacity for regeneration and result in heightened inflammation and fibrotic tissue deposition resulting in loss of structure and function. Recent studies have focused on biomaterial and stem cell-based therapies to promote skeletal muscle regeneration following injury and disease. Many stem cell populations besides satellite cells are implicated in muscle regeneration. These stem cells include but are not limited to mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells, pericytes, fibroadipogenic progenitors, side population cells, and CD133+ stem cells. However, several challenges associated with their isolation, availability, delivery, survival, engraftment, and differentiation have been reported in recent studies. While acellular scaffolds offer a relatively safe and potentially off-the-shelf solution to cell-based therapies, they are often unable to stimulate host cell migration and activity to a level that would result in clinically meaningful regeneration of traumatized muscle. Combining stem cells and biomaterials may offer a viable therapeutic strategy that may overcome the limitations associated with these therapies when they are used in isolation. In this article, we review the stem cell populations that can stimulate muscle regeneration in vitro and in vivo. We also discuss the regenerative potential of combination therapies that utilize both stem cell and biomaterials for the treatment of skeletal muscle injury and disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1246-1262, 2019.

Biomimetic sponges for regeneration of skeletal muscle following trauma.

Skeletal muscle is inept in regenerating after traumatic injuries due to significant loss of basal lamina and the resident satellite cells. To improve regeneration of skeletal muscle, we have developed biomimetic sponges composed of collagen, gelatin, and laminin (LM)-111 that were crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Collagen and LM-111 are crucial components of the muscle extracellular matrix and were chosen to impart bioactivity whereas gelatin and EDC were used to provide mechanical strength to the scaffold. Morphological and mechanical evaluation of the sponges showed porous structure, water-retention capacity and a compressive modulus of 590-808 kPa. The biomimetic sponges supported the infiltration and viability of C2 C12 myoblasts over 5 days of culture. The myoblasts produced higher levels of myokines such as VEGF, IL-6, and IGF-1 and showed higher expression of myogenic markers such as MyoD and myogenin on the biomimetic sponges. Biomimetic sponges implanted in a mouse model of volumetric muscle loss (VML) supported satellite, endothelial, and inflammatory cell infiltration but resulted in limited myofiber regeneration at 2 weeks post-injury. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 92-103, 2019.

The Effect of Laminin-111 Hydrogels on Muscle Regeneration in a Murine Model of Injury.

IMPACT STATEMENT: Extremity injuries make up the most common survivable injuries in vehicular accidents and modern military conflicts. A majority of these injuries involve volumetric muscle loss (VML). The potential for donor site morbidity may limit the clinical use of autologous muscle grafts for VML injuries. Treatments that can improve the regeneration of functional muscle tissue are critically needed to improve limb salvage and reduce the rate of delayed amputations. The development of a laminin-111-enriched fibrin hydrogel will offer a potentially transformative and "off-the-shelf" clinically relevant therapy for functional skeletal muscle regeneration.

Laminin-111 functionalized polyethylene glycol hydrogels support myogenic activity in vitro.

Skeletal muscle has a remarkable regenerative capability following mild physical or chemical insult. However, following a critical loss of muscle tissue, the regeneration process is impaired due to the inadequate myogenic activity of muscle resident stem cells (i.e., satellite cells). Laminin (LM) is a heterotrimeric structural protein in the satellite cell niche that is crucial for maintaining its function. In this study, we created hydrogels composed of poly (ethylene glycol) (PEG) and LM-111 to provide an elastic substrate for satellite cell proliferation at the site of injury. The PEG-LM111 conjugates were mixed with 5% and 10% (w/v) pure PEG-diacrylate (PEGDA) and photopolymerized to form 5% and 10% PEGLM gels. Pure 5% and 10% PEGDA gels were used as controls. The modulus of both hydrogels containing 10% (w/v) PEGDA was significantly higher than the hydrogels containing 5% (w/v) PEGDA. The 5% PEGLM hydrogels showed significantly higher swelling in aqueous medium suggesting a more porous structure. C2C12 myoblasts cultured on the softer 5% PEGLM hydrogels showed a flat and spread-out morphology when compared to the rounded, multicell clusters formed on the 5% PEGDA, 10% PEGDA, and 10% PEGLM hydrogels. The 5% PEGLM hydrogels also promoted a significant increase in both vascular endothelial growth factor and interleukin-6 (IL-6) production from the myoblasts. Additionally, the expression of MyoD was significantly higher while that of myogenin and α-actinin trended higher on the 5% PEGLM hydrogels compared to 5% PEGDA on day 5. Our data suggests that the introduction of LM-111 into compliant PEG hydrogels promoted myoblast adhesion, survival, pro-regenerative growth factor production, and myogenic activity.

Laminin-111 enriched fibrin hydrogels for skeletal muscle regeneration.

Laminin (LM)-111 supplementation has improved muscle regeneration in several models of disease and injury. This study investigated a novel hydrogel composed of fibrinogen and LM-111. Increasing LM-111 concentration (50-450 µg/mL) in fibrin hydrogels resulted in highly fibrous scaffolds with progressively thinner interlaced fibers. Rheological testing showed that all hydrogels had viscoelastic behavior and the Young's modulus ranged from 2-6KPa. C2C12 myobalsts showed a significant increase in VEGF production and decrease in IL-6 production on LM-111 enriched fibrin hydrogels as compared to pure fibrin hydrogels on day 4. Western blotting results showed a significant increase in MyoD and desmin protein quantity but a significant decrease in myogenin protein quantity in myoblasts cultured on the LM-111 (450 µg/mL) enriched fibrin hydrogel. Combined application of electromechanical stimulation significantly enhanced the production of VEGF and IGF-1 from myoblast seeded fibrin-LM-111 hydrogels. Taken together, these observations offer an important first step toward optimizing a tissue engineered constructs for skeletal muscle regeneration.