Comparing electrical stimulation and tacrolimus (FK506) to enhance treating nerve injuries.

INTRODUCTION: Neuroenhancing therapies are desired because repair of nerve injuries can fail to achieve recovery. We compared two neuroenhancing therapies, electrical stimulation (ES) and systemic tacrolimus (FK506), for their capabilities to enhance regeneration in the context of a rat model. METHODS: Rats were randomized to four groups: ES 0.5 mA, ES 2.0 mA, FK506, and repair alone. All groups underwent tibial nerve transection and repair, and outcomes were assessed by using twice per week walking track analysis, cold allodynia response, relative muscle mass, and nerve histology. RESULTS: Electrical stimulation and FK506 groups demonstrated improved functional recovery and myelinated axon counts distal to the repair compared with repair alone. Electrical stimulation provided improvements in nerve regeneration that were not different from optimized FK506 systemic administration. DISCUSSION: Providing ES after nerve repair improved regeneration and recovery in rats, with minimal differences in therapeutic efficacy to FK506, further demonstrating its clinical potential to improve management of nerve injuries.

Effects of connective tissue growth factor on the regulation of elastogenesis in human umbilical cord-derived mesenchymal stem cells.

BACKGROUND: A key to clinical microtia reconstruction is construct flexibility. The most significant current limitation to engineered elastic cartilage is maintaining an elastic phenotype, which is principally dependent on elastin production (although other parameters, including maintenance of a ratio above 1 for collagens II to I, minimizing collagen X content, and presence of adequate matrix fibrillin for elastin binding, all play supporting roles). Connective tissue growth factor (CTGF), a compound secreted by chondrocytes, has been shown to promote an elastic phenotype in mature rabbit chondrocytes; however, CTGF effect on undifferentiated mesenchymal stem cells (MSCs) has not been characterized. The principal aim of this study is to analyze CTGF effect on elastin production in umbilical cord (UC)-derived MSCs and to determine optimal timing of treatment to maximize elastin production. METHODS: Human UCMSCs (hUCMSCs) were isolated from Wharton jelly using an explant technique, grown to passage 3, seeded onto nanofiber scaffolds, and chondroinduced for 21 days. Nanofiber scaffolds were electrospun using solubilized poly L-lactide/D-lactide/glycolide (PLGA). Chondrogenic media was supplemented with 25 µg/mL CTGF starting at day 0 or 7. Messenger RNA (mRNA) for Collagen I, II, X, fibrillin, and elastin was quantified by RT-PCR; glycosaminoglycan (GAG) matrix deposition was assessed and normalized by cellular DNA content. Elastin protein was assessed by Western blot analysis. All experiments were performed in triplicate with MSCs from 4 distinct cords. Multiway analysis of variance with Newman-Keuls post test was used to determine statistical significance. RESULTS: Connective tissue growth factor treatment results in increased GAG/DNA ratio; the differentiation index was maintained above 1 in all conditions, with increased collage II noted at days 7 and 14 in CTGF conditions; no difference in collagen X or fibrillin mRNA was noted. Increased elastin mRNA and protein were noted at day 14 in conditions treated with CTGF at day 7 after differentiation. CONCLUSIONS: Connective tissue growth factor leads to maximal elastin increase in UCMSCs after 7 days of chondroinduction and not in undifferentiated MSCs. With appropriately timed treatment, CTGF may be a useful adjunct in maintaining an elastic cartilage phenotype in engineered cartilage from human UCMSCs.

Profiling the molecular signature of satellite glial cells at the single cell level reveals high similarities between rodents and humans.

ABSTRACT: Peripheral sensory neurons located in dorsal root ganglia relay sensory information from the peripheral tissue to the brain. Satellite glial cells (SGCs) are unique glial cells that form an envelope completely surrounding each sensory neuron soma. This organization allows for close bidirectional communication between the neuron and its surrounding glial coat. Morphological and molecular changes in SGC have been observed in multiple pathological conditions such as inflammation, chemotherapy-induced neuropathy, viral infection, and nerve injuries. There is evidence that changes in SGC contribute to chronic pain by augmenting the neuronal activity in various rodent pain models. Satellite glial cells also play a critical role in axon regeneration. Whether findings made in rodent model systems are relevant to human physiology have not been investigated. Here, we present a detailed characterization of the transcriptional profile of SGC in mice, rats, and humans at the single cell level. Our findings suggest that key features of SGC in rodent models are conserved in humans. Our study provides the potential to leverage rodent SGC properties and identify potential targets in humans for the treatment of nerve injuries and alleviation of painful conditions.

T cells modulate IL-4 expression by eosinophil recruitment within decellularized scaffolds to repair nerve defects.

Decellularized nerve, or acellular nerve allografts (ANAs), are an increasingly used alternative to nerve autografts to repair nerve gaps to facilitate regeneration. The adaptive immune system, specifically T cells, plays a role in promoting regeneration upon these ANA scaffolds. However, how T cells promote regeneration across ANAs is not clear. Here, we show that T cells accumulate within ANAs repairing nerve gaps resulting in regulation of cytokine expression within the ANA environment. This in turn ultimately leads to robust nerve regeneration and functional recovery. Nerve regeneration across ANAs and functional recovery in Rag1KO mice was limited compared to wild-type (WT) mice. Prior to appreciable nerve regeneration, ANAs from Rag1KO mice contained fewer eosinophils and reduced IL-4 expression compared to ANAs from WT mice. During this period, both T cells and eosinophils regulated IL-4 expression within ANAs. Eosinophils represented the majority of IL-4 expressing cells within ANAs, while T cells regulated IL-4 expression. Finally, an essential role for IL-4 during nerve regeneration across ANAs was confirmed as nerves repaired using ANAs had reduced regeneration in IL-4 KO mice compared to WT mice. Our data demonstrate T cells regulate the expression of IL-4 within the ANA environment via their effects on eosinophils. STATEMENT OF SIGNIFICANCE: The immune system has been emerging as a critical component for tissue regeneration, especially when regeneration is supported upon biomaterials. The role of T cells, and their roles in the regeneration of nerve repaired with biomaterials, is still unclear. We demonstrated that when nerves are repaired with decellularized nerve scaffolds, T cells contribute to regeneration by regulating cytokines. We focused on their regulation of cytokine IL-4. Unexpectedly, T cells do not produce IL-4, but instead regulate IL-4 by recruiting eosinophils, which are major cellular sources of IL-4 within these scaffolds. Thus, our work demonstrated how IL-4 is regulated in a model biomaterial, and has implications for improving the design of biomaterials and understanding immune responses to biomaterials.

A Versatile Synthetic Extracellular Matrix Mimic via Thiol-Norbornene Photopolymerization.

Step-growth, radically mediated thiol-norbornene photopolymerization is used to create versatile, stimuli-responsive poly(ethylene glycol)-co-peptide hydrogels The reaction is cytocompatible and allows for the encapsulation of human mesenchymal stem cells with a viability greater than 95%. Cellular spreading is dictated via three-dimensional biochemical photopatterning.

Nociceptor Deletion of Tsc2 Enhances Axon Regeneration by Inducing a Conditioning Injury Response in Dorsal Root Ganglia.

Neurons of the PNS are able to regenerate injured axons, a process requiring significant cellular resources to establish and maintain long-distance growth. Genetic activation of mTORC1, a potent regulator of cellular metabolism and protein translation, improves axon regeneration of peripheral neurons by an unresolved mechanism. To gain insight into this process, we activated mTORC1 signaling in mouse nociceptors via genetic deletion of its negative regulator Tsc2. Perinatal deletion of Tsc2 in nociceptors enhanced initial axon growth after sciatic nerve crush, however by 3 d post-injury axon elongation rate became similar to controls. mTORC1 inhibition prior to nerve injury was required to suppress the enhanced axon growth. Gene expression analysis in purified nociceptors revealed that Tsc2-deficient nociceptors had increased activity of regeneration-associated transcription factors (RATFs), including cJun and Atf3, in the absence of injury. Additionally, nociceptor deletion of Tsc2 activated satellite glial cells and macrophages in the dorsal root ganglia (DRG) in a similar manner to nerve injury. Surprisingly, these changes improved axon length but not percentage of initiating axons in dissociated cultures. The pro-regenerative environment in naïve DRG was recapitulated by AAV8-mediated deletion of Tsc2 in adult mice, suggesting that this phenotype does not result from a developmental effect. Consistently, AAV8-mediated Tsc2 deletion did not improve behavioral recovery after a sciatic nerve crush injury despite initially enhanced axon growth. Together, these data show that neuronal mTORC1 activation induces an incomplete pro-regenerative environment in the DRG that improves initial but not later axon growth after nerve injury.

Predictors of Dependency in Geriatric Trauma Patients with Rib Fractures: A Population Study.

The geriatric trauma population is unique. These patients are at risk of being discharged to rehabilitation or a skilled nursing facility, instead of being returned to their homes, placing a significant burden on both the patient families and society. This study evaluated which patient characteristics increase the likelihood of a previously independent geriatric blunt trauma becoming functionally dependent and being discharged to a location other than home. Data were extracted from the National Trauma Data Bank from 2012 to 2014 for blunt trauma patients ≥65 years old, admitted from home, with one or more rib fractures. Primary outcomes were discharge home versus a facility. Subgroup analysis evaluated disposition to acute short-term rehabilitation or subacute rehabilitation or skilled nursing facility. Multivariable analysis was used to calculate probabilities of disposition based on the above variables, controlling for comorbidities. Sixteen thousand six hundred thirty-two patients were included. Only 58 per cent were discharged home. Increased age, ≥4 rib fractures, white race, and female gender were found to increase the risk of discharge to a facility. In addition, patients with chronic renal failure, history of diabetes, obesity, or heart failure were less likely to be discharged home. This study shows that age, gender, race, and the number of rib fractures are statistically significant in predicting which patients are less likely to be discharged home. This reinforces the need for the development of triage and treatment protocols in this higher risk population, to decrease the social and financial burden of these injuries.

A pilot study comparing mechanical properties of tissue-engineered cartilages and various endogenous cartilages.

BACKGROUND: Mechanical properties of tissue-engineered cartilage and a variety of endogenous cartilage were measured. The main goal was to evaluate if the tissue-engineered cartilage have similar mechanical characteristics to be replaced with rib cartilage in microtia reconstruction. Such study lays the foundation for future human clinical trials for microtia reconstruction. METHOD: Atomic force microscopy and compression testing were used to measure the viscoelasticity of tissue-engineered cartilage (stem cell seeded on Poly lactic co-glycolytic acid nanofibers and Pellet) and endogenous cartilage: conchal bowl, microtic ears, preauricular remnants, and rib. Atomic force microscopy, calculates biomaterial elasticity through force-deformation measurement and Hertz model. Compression testing determines the stress relaxation by measuring slope of stress reduction at 10% strain. FINDING: Tissue-engineered cartilage demonstrated elasticity (4.6kPa for pellet and 6.6kPa for PLGA) and stress relaxation properties (7.6 (SD 1.1) kPa/s for pellet) most similar to those of native conchal bowl cartilage (31.8 (SD 18) kPa for the elasticity and 15.1 (SD 2.1) kPa/s for stress relaxation factor). Rib cartilage was most dissimilar from the mechanical characteristics of conchal cartilage and demonstrated the highest elastic modulus (361 (SD 372) kPa). Moreover, except preauricular cartilage samples, the level of elastic modulus increased with age. INTERPRETATION: The use of tissue-engineered cartilage developed via PLGA and Pellet methods, may be an appropriate substitute for rib cartilage in the reconstruction of microtic ears, however their mechanical characteristics still need to be improved and require further validation in animal studies.

Giant type III well-differentiated neuroendocrine tumor of the stomach: A case report.

INTRODUCTION: We describe a case of a large type III neuroendocrine tumor of the stomach. Management and current literature are reviewed. PRESENTATION OF CASE: A 37year old female presented with upper gastrointestinal bleed and epigastric pain. Further workup demonstrated a large ulcerated gastric mass near the GE junction. Computer tomography scan and endoscopic ultrasound showed a 10cm mass with no evidence of distant disease. Fine needle aspiration pathology was consistent with a well differentiated neuroendocrine tumor (Ki67 index<2%), with elevated levels of chromogranin A and serotonin levels but normal gastrin. The patient underwent an uneventful total gastrectomy. Final pathology analysis reported a higher KI67 index (7.54%) and a final pathology of grade 2 type III, T3 N3, neuroendocrine tumor of the stomach. The chromogranin levels normalized and no recurrent disease has been detected in one year follow up. DISCUSSION: Gastric neuroendocrine tumors are extremely rare, accounting for 4% of all neuroendocrine tumors of the body and 1% of all neoplasms of the stomach. Based on histomorphologic characteristics and pathogenesis, gastric neuroendocrine tumors are classified into four types with differing prognosis and behavior. Current literature describes type 3 gastric neuroendocrine tumors as larger than 2cm. However, there is no precedent in the literature for a tumor of this size. CONCLUSION: The incidence of gastric neuroendocrine tumors has been increasing during the last decade, underscoring the need to improve our understanding of their biology and behavior. When identified histologically, patient outcomes depend on appropriate determination of tumor biology and subsequent choice of treatment.

Juvenile Swine Surgical Alveolar Cleft Model to Test Novel Autologous Stem Cell Therapies.

Reconstruction of craniofacial congenital bone defects has historically relied on autologous bone grafts. Engineered bone using mesenchymal stem cells from the umbilical cord on electrospun nanomicrofiber scaffolds offers an alternative to current treatments. This preclinical study presents the development of a juvenile swine model with a surgically created maxillary cleft defect for future testing of tissue-engineered implants for bone generation. Five-week-old pigs (n=6) underwent surgically created maxillary (alveolar) defects to determine critical-sized defect and the quality of treatment outcomes with rib, iliac crest cancellous bone, and tissue-engineered scaffolds. Pigs were sacrificed at 1 month. Computed tomography scans were obtained at days 0 and 30, at the time of euthanasia. Histological evaluation was performed on newly formed bone within the surgical defect. A 1 cm surgically created defect healed with no treatment, the 2 cm defect did not heal. A subsequently created 1.7 cm defect, physiologically similar to a congenitally occurring alveolar cleft in humans, from the central incisor to the canine, similarly did not heal. Rib graft treatment did not incorporate into adjacent normal bone; cancellous bone and the tissue-engineered graft healed the critical-sized defect. This work establishes a juvenile swine alveolar cleft model with critical-sized defect approaching 1.7 cm. Both cancellous bone and tissue engineered graft generated bridging bone formation in the surgically created alveolar cleft defect.