Identifying dysregulated immune cell subsets following volumetric muscle loss with pseudo-time trajectories.

Volumetric muscle loss (VML) results in permanent functional deficits and remains a substantial regenerative medicine challenge. A coordinated immune response is crucial for timely myofiber regeneration, however the immune response following VML has yet to be fully characterized. Here, we leveraged dimensionality reduction and pseudo-time analysis techniques to elucidate the cellular players underlying a functional or pathological outcome as a result of subcritical injury or critical VML in the murine quadriceps, respectively. We found that critical VML resulted in a sustained presence of M2-like and CD206hiLy6Chi 'hybrid' macrophages whereas subcritical defects resolved these populations. Notably, the retained M2-like macrophages from critical VML injuries presented with aberrant cytokine production which may contribute to fibrogenesis, as indicated by their co-localization with fibroadipogenic progenitors (FAPs) in areas of collagen deposition within the defect. Furthermore, several T cell subpopulations were significantly elevated in critical VML compared to subcritical injuries. These results demonstrate a dysregulated immune response in critical VML that is unable to fully resolve the chronic inflammatory state and transition to a pro-regenerative microenvironment within the first week after injury. These data provide important insights into potential therapeutic strategies which could reduce the immune cell burden and pro-fibrotic signaling characteristic of VML.

Increased fluorescence observation intensity during the photodynamic diagnosis of deeply located tumors by fluorescence photoswitching of protoporphyrin IX.

Significance: Photobleaching of the photosensitizer reduces fluorescence observation time and the intensity of fluorescence emitted for tumor detection during 5-aminolevulinic acid-based photodynamic diagnosis. Aim: This study aims to utilize the concept of fluorescence photoswitching, which uses the fluorescence emission from photosensitizer excitation followed by the simultaneous excitation of the photosensitizer and its photoproduct to increase the fluorescence detection intensity during PDD of deeply located tumors. Approach: The fluorescence photobleaching of protoporphyrin IX (PpIX) and the formation of its photoproduct, photoprotoporhyrin (Ppp), caused by exposure to 505 nm light were investigated in solution, ex vivo, and in vivo, and the fluorescence photoswitching was analyzed. The fluorescence observations of PpIX and Ppp were performed with 505 and 450 or 455 nm excitation, respectively, which is the suited wavelength for the primary excitation of each fluorophore. Results: Fluorescence photoswitching was observed in all forms of PpIX investigated, and the fluorescence photoswitching time, fluorescence intensity relative to the initial PpIX and Ppp intensity, and fluorescence intensity relative to PpIX after photobleaching were obtained. The dependence of the fluorescence photoswitching time and intensity on the irradiation power density was noted. A fluorescence intensity increase between 1.6 and 3.9 times was achieved with simultaneous excitation of PpIX and Ppp after fluorescence photoswitching, compared with the excitation of PpIX alone. Conclusions: We have demonstrated the potential of fluorescence photoswitching for the improvement of the fluorescence observation intensity for the PDD of deeply located tumors.

Nanofiber-Based Delivery of Bioactive Lipids Promotes Pro-regenerative Inflammation and Enhances Muscle Fiber Growth After Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries after extremity trauma results in an important clinical challenge often associated with impaired healing, significant fibrosis, and long-term pain and functional deficits. While acute muscle injuries typically display a remarkable capacity for regeneration, critically sized VML defects present a dysregulated immune microenvironment which overwhelms innate repair mechanisms leading to chronic inflammation and pro-fibrotic signaling. In this series of studies, we developed an immunomodulatory biomaterial therapy to locally modulate the sphingosine-1-phosphate (S1P) signaling axis and resolve the persistent pro-inflammatory injury niche plaguing a critically sized VML defect. Multiparameter pseudo-temporal 2D projections of single cell cytometry data revealed subtle distinctions in the altered dynamics of specific immune subpopulations infiltrating the defect that were critical to muscle regeneration. We show that S1P receptor modulation via nanofiber delivery of Fingolimod (FTY720) was characterized by increased numbers of pro-regenerative immune subsets and coincided with an enriched pool of muscle stem cells (MuSCs) within the injured tissue. This FTY720-induced priming of the local injury milieu resulted in increased myofiber diameter and alignment across the defect space followed by enhanced revascularization and reinnervation of the injured muscle. These findings indicate that localized modulation of S1P receptor signaling via nanofiber scaffolds, which resemble the native extracellular matrix ablated upon injury, provides great potential as an immunotherapy for bolstering endogenous mechanisms of regeneration following VML injury.

Modulating local S1P receptor signaling as a regenerative immunotherapy after volumetric muscle loss injury.

Regeneration of skeletal muscle after volumetric injury is thought to be impaired by a dysregulated immune microenvironment that hinders endogenous repair mechanisms. Such defects result in fatty infiltration, tissue scarring, chronic inflammation, and debilitating functional deficits. Here, we evaluated the key cellular processes driving dysregulation in the injury niche through localized modulation of sphingosine-1-phosphate (S1P) receptor signaling. We employ dimensionality reduction and pseudotime analysis on single cell cytometry data to reveal heterogeneous immune cell subsets infiltrating preclinical muscle defects due to S1P receptor inhibition. We show that global knockout of S1P receptor 3 (S1PR3) is marked by an increase of muscle stem cells within injured tissue, a reduction in classically activated relative to alternatively activated macrophages, and increased bridging of regenerating myofibers across the defect. We found that local S1PR3 antagonism via nanofiber delivery of VPC01091 replicated key features of pseudotime immune cell recruitment dynamics and enhanced regeneration characteristic of global S1PR3 knockout. Our results indicate that local S1P receptor modulation may provide an effective immunotherapy for promoting a proreparative environment leading to improved regeneration following muscle injury.