Intramuscular Nanofat Injection Promotes Inflammation-Induced Gastrocnemius Regeneration in a Syngeneic Rat Sciatic Nerve Injury Model.

BACKGROUND: Mechanical emulsification of adipose tissue to concentrate protein and stromal cell components (ie, nanofat) has gained considerable interest in clinical practice. Although the regenerative potential of nanofat has largely been used in aesthetic applications, these effects have considerable potential in reconstruction as well. Here, the authors investigated the therapeutic properties of nanofat injected directly into the denervated gastrocnemius after a sciatic nerve injury in Lewis rats. METHODS: Muscle denervation was induced by transecting and immediately repairing the sciatic nerve. Inguinal and subcutaneous adipose was harvested from donor rodents, processed into nanofat, and then injected intramuscularly into the gastrocnemius. Gait analysis was performed weekly. Rodents were euthanized at 9 and 12 weeks, after which tetanic contraction force was measured, and gene expression, histology, and cytokine multiplexing were performed. RESULTS: Intramuscular injection of nanofat significantly increased maximum tetanic force generation at 9 and 12 weeks. The forces of the nanofat-injected gastrocnemii were better correlated to their contralateral gastrocnemii relative to controls. Muscle repair-associated inflammatory gene expressions were significantly up-regulated in nanofat-injected gastrocnemii. Cytokines interleukin (IL)-1ß, IL-18, vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, and tissue inhibitor of metalloproteinase-1 were significantly higher in nanofat-injected gastrocnemii relative to control gastrocnemii, and the tetanic force was linearly and significantly correlated to IL-1ß and IL-18 and their interacting effects. CONCLUSIONS: Intramuscular injection of emulsified adipose tissue (nanofat) significantly increased gastrocnemii contraction force after sciatic nerve injury, with prolonged reconstructive inflammation by means of CD68, inducible nitric oxide synthase, IL-1ß, and IL-18 all being potential mechanisms for this recovery. This application could potentially increase the therapeutic breadth of nanofat to include muscular recovery after nerve injury. CLINICAL RELEVANCE STATEMENT: The authors' study investigates a clinically translatable therapy to mitigate muscle atrophy after nerve injury.

Biodegradable Nerve Guide with Glial Cell Line-Derived Neurotrophic Factor Improves Recovery After Facial Nerve Injury in Rats.

Background: Bioengineered nerve guides with glial cell line-derived neurotrophic factor (GDNF) support recovery after facial nerve injury by acting as regenerative scaffolds. Objective: To compare functional, electrophysiological, and histological outcomes after repair of rat facial nerve transection in control, empty nerve guide, and nerve guide with GDNF conditions. Methods: Rats underwent transection and primary repair of the buccal branch of the facial nerve and were divided into (1) transection and repair only, (2) transection and repair augmented with empty guide, (3) transection and repair augmented with GDNF-guide groups. Weekly measurements of the whisking movements were recorded. At 12 weeks, compound muscle action potentials (CMAPs) at the whisker pad were assessed, and samples were collected for histomorphometric analysis. Results: Rats in GDNF-guide group displayed the earliest peak in normalized whisking amplitude. CMAPs were significantly higher after GDNF-guide placement. Mean fiber surface area of the target muscle, axonal count of the injured branch, and the number of Schwann cells were highest with GDNF guides. Conclusion: The biodegradable nerve guide containing double-walled GDNF microspheres enhanced recovery after facial nerve transection and primary repair.

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery. The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Intramuscular injection of skeletal muscle derived extracellular matrix mitigates denervation atrophy after sciatic nerve transection.

Peripheral nerve injury and the associated muscle atrophy has an estimated annual healthcare burden of $150 billion dollars in the United States. When considering the total annual health-related spending of $3.5 trillion, these pathologies alone occupy about 4.3%. The prevalence of these ailments is rooted, at least in part, in the lack of specific preventative therapies that can be administered to muscle while it remains in the denervated state. To address this, skeletal muscle-derived ECM (skECM) was injected directly in denervated muscle with postoperative analysis performed at 20 weeks, including gait analysis, force production, cytokine quantification, and histological analysis. skECM was shown to be superior against non-injected muscle controls showing no difference in contraction force to uninjured muscle at 20 weeks. Cytokines IL-1ß, IL-18, and IFNγ appeared to mediate regeneration with statistical regression implicating these cytokines as strong predictors of muscle contraction, showing significant linear correlation.

Design and Fabrication of an Automatable, 3D Printed Perfusion Device for Tissue Infusion and Perfusion Engineering.

Tissue decellularization for generating extracellular matrices has become a staple of regenerative medicine in the recent decades, extending from the research setting to clinical usage. Although methods and protocols for tissue decellularization are abundant throughout the literature, they can be time intensive and typically require specific overhead in terms of equipment. To reduce these barriers to entry, a functional and reproducible prototype of a tissue infusion/perfusion device (TIPD) has been designed and fabricated using three-dimensional printed parts in conjunction with commercially available components. This TIPD forms a system composed of two peristaltic pumps, two 3-way valves, and a chamber in which tissue is contained, and is controlled by user-customizable software. To increase repeatability among decellularization protocols, an automation function has been integrated into the software, which is able to specify fluid flow rates and define specific valve locations enabling selection of solutions to be introduced into a scaffold over the course of a decellularization process. The prototype has been tested for proof of concept through infusion and perfusion decellularization of skeletal muscle and intact kidneys, respectively, and has shown successful removal of cellular content while maintaining an intact ultrastructure. In an effort to increase the reproducibility of experimental designs and to promote an open source hardware initiative in the field of tissue engineering, a novel device was conceptualized and prototyped with printable part files made available for its fabrication in tandem with instructions for assembly. Impact Statement Repeatable methods for decellularization are essential for achieving consistent substrates between batches, laboratories, and facilities. To meet this end, an automatable tissue infusion/perfusion device composed of three-dimensional printed parts and commercially available components has been prototyped and tested. Materials and instructions for its assembly have been made available in an effort to reduce variability among equipment as well as to provide a platform on which to iterate open-source hardware in tissue engineering.