SCAN for Abuse: Electronic Health Record-Based Universal Child Abuse Screening.

BACKGROUND: Identification of physical abuse at the point of care without a systematic approach remains inherently subjective and prone to judgement error. This study examines the implementation of an electronic health record (EHR)-based universal child injury screen (CIS) to improve detection rates of child abuse. METHODS: CIS was implemented in the EHR admission documentation for all patients age 5 or younger at a single medical center, with the following questions. 1) "Is this patient an injured/trauma patient?" 2) "If this is a trauma/injured patient, where did the injury occur?" A "Yes" response to Question 1 would alert a team of child abuse pediatricians and social workers to determine if a patient required formal child abuse clinical evaluation. Patients who received positive CIS responses, formal child abuse work-up, and/or reports to Child Protective Services (CPS) were reviewed for analysis. CPS rates from historical controls (2017-2018) were compared to post-implementation rates (2019-2021). RESULTS: Between 2019 and 2021, 14,150 patients were screened with CIS. 286 (2.0 %) patients screened received positive CIS responses. 166 (58.0 %) of these patients with positive CIS responses would not have otherwise been identified for child abuse evaluation by their treating teams. 18 (10.8 %) of the patients identified by the CIS and not by the treating team were later reported to CPS. Facility CPS reporting rates for physical abuse were 1.2 per 1000 admitted children age 5 or younger (pre-intervention) versus 4.2 per 1000 (post-intervention). CONCLUSIONS: Introduction of CIS led to increased detection suspected child abuse among children age 5 or younger. LEVEL OF EVIDENCE: Level II. TYPE OF STUDY: Study of Diagnostic Test.

Complications Associated with Subsequent Vascular Access in Pediatric ECMO Patients.

INTRODUCTION: Following ECMO decannulation, intensivists and surgeons must consider whether to reuse the cannulation site for central venous catheters (CVC) or seek remote access. This study investigates the risk of infectious complication associated with the reuse of peripheral ECMO cannulation sites for subsequent central venous access. METHODS: A retrospective review was conducted for patients aged 0-18 years, who underwent peripheral ECMO cannulation between 2009 and 2021 at a single children's hospital. RESULTS: Of the 227 charts reviewed, after ECMO decannulation, 53 patients received a CVC at the same location, 25 received a CVC at a different location, 62 received a peripherally inserted central catheter (PICC), and 87 had no subsequent vascular access placed within 30 days of decannulation. Patients with secondary access placed at the same site experienced 1 CLABSI, or 0.94 CLABSIs per 1000 line days. Patients with PICC lines after ECMO decannulation had 1 CLABSI, or 0.43 CLABSIs per 1000 line days. In comparison, the institution's hospital-wide CLABSI rate was 1.46 per 1000 line days during this same period. Although the rate of CLABSI among patients with secondary access at the site of decannulation was higher than the rate among patients with PICC lines (p = 0.79) it was lower than the institutional rate (p = 0.54), these differences did not rise to the level of statistical significance. CONCLUSION: Compared with ECMO patients with subsequent CVCs placed at an alternative access site or via PICC after decannulation, patients with contemporaneous CVC placement at the site of decannulation do not experience a significantly higher rate of CLABSIs. LEVEL OF EVIDENCE: Level III. TYPE OF STUDY: Retrospective comparative study.

Sustained Release of Tacrolimus From a Topical Drug Delivery System Promotes Corneal Reinnervation.

Purpose: Corneal nerve fibers provide sensation and maintain the epithelial renewal process. Insufficient corneal innervation can cause neurotrophic keratopathy. Here, topically delivered tacrolimus is evaluated for its therapeutic potential to promote corneal reinnervation in rats. Methods: A compartmentalized neuronal cell culture was used to determine the effect of locally delivered tacrolimus on sensory axon regeneration in vitro. The regenerating axons but not the cell bodies were exposed to tacrolimus (50 ng/mL), nerve growth factor (50 ng/mL), or a vehicle control. Axon area and length were measured after 48 hours. Then, a biodegradable nanofiber drug delivery system was fabricated via electrospinning of a tacrolimus-loaded polycarbonate-urethane polymer. Biocompatibility, degradation, drug biodistribution, and therapeutic effectiveness were tested in a rat model of neurotrophic keratopathy induced by stereotactic trigeminal nerve ablation. Results: Sensory neurons whose axons were exposed to tacrolimus regenerated significantly more and longer axons compared to vehicle-treated cultures. Trigeminal nerve ablation in rats reliably induced corneal denervation. Four weeks after denervation, rats that had received tacrolimus topically showed similar limbal innervation but a significantly higher nerve fiber density in the center of the cornea compared to the non-treated control. Topically applied tacrolimus was detectable in the ipsilateral vitreal body, the plasma, and the ipsilateral trigeminal ganglion but not in their contralateral counterparts and vital organs after 4 weeks of topical release. Conclusions: Locally delivered tacrolimus promotes axonal regeneration in vitro and corneal reinnervation in vivo with minimal systemic drug exposure. Translational Relevance: Topically applied tacrolimus may provide a readily translatable approach to promote corneal reinnervation.

Early Discontinuation, Results Reporting, and Publication of Gynecology Clinical Trials From 2007 to 2020.

OBJECTIVE: To characterize gynecology clinical trials over time, compare gynecology subspecialties, and analyze factors associated with early discontinuation, results reporting, and publication. METHODS: We conducted a cross-sectional analysis of all gynecology trials registered on ClinicalTrials.gov between 2007 and 2020 and their resulting publications. Trials were analyzed with descriptive, multivariable logistic, and Cox regression analyses. Primary exposure variables were trial funding and subspecialty. The three primary outcomes included early discontinuation, results reporting to ClinicalTrials.gov, and publication in a peer-reviewed journal indexed on PubMed. RESULTS: Of 223,690 trials registered on ClinicalTrials.gov between October 2007 and March 2020, only 3.7% focused on gynecology (n=8,174, approximately 3,759,086 participants). Subspecialties included reproductive endocrinology and infertility (n=1,428, 17.5%), gynecologic oncology (n=2,063, 25.2%), urogynecology (n=1,118, 13.7%), family planning (n=648, 7.9%), and other benign gynecology (n=2,917, 35.7%). Only 42.0% of completed trials disseminated results through results reporting and publication. Of all funding types, industry-funded trials were the most likely to be discontinued early (P<.001). Academic-funded trials were the least likely to report results (adjusted odds ratio [aOR] 0.38, 95% CI 0.30-0.50) but the most likely to publish (aOR 1.62, 95% CI 1.24-2.12). The number of reproductive endocrinology and infertility trials increased the most of any subspecialty between 2007 and 2020 (6.4% growth rate). Reproductive endocrinology and infertility and family planning trials were the most likely to be stopped early (reproductive endocrinology and infertility: adjusted hazard ratio [aHR] 2.08, 95% CI 1.59-2.71; family planning: aHR 1.55 95% CI 1.06-2.25). When completed, reproductive endocrinology and infertility trials were the least likely to report results (aOR 0.58, 95% CI 0.38-0.88). No significant differences were seen between subspecialties with respect to publication. CONCLUSION: Gynecology trials comprise only 3.7% of all clinical trials. The paucity of gynecology clinical trials aligns with decades of female underrepresentation in research. When completed, gynecology trials have poor dissemination. Our findings raise concern about bias in the performance, reporting, and publication of gynecology clinical trials.

Mental health outcomes in pediatric trauma patients: A 10 year real world analysis using a large database approach.

INTRODUCTION: Traumatic injury is the leading cause of morbidity and mortality among children in the United States. Single institution studies suggest an increased risk of poor mental health outcomes among these patients, but there are few population-based studies assessing this risk. METHODS: The IBMⓇ MarketScanⓇ private insurance claims database was used to identify children (6-17yo) with traumatic injuries between 2007 and 2016. Time-to-event analysis was performed to compare rates of PTSD, depression, anxiety, and adjustment disorder among children admitted to the hospital compared to children treated in the emergency department (ED), urgent care (UC), or in the outpatient setting, and to children admitted with uncomplicated appendicitis. RESULTS: Among children admitted for traumatic injury, 3.3% developed a subsequent mental health diagnosis, and 1.6% developed PTSD. Children admitted for traumatic injury were at increased risk of developing a mental health condition (HR 1.34, p < 0.001) compared to those admitted for appendicitis. Children treated in the ED or UC for traumatic injury and those treated in the outpatient setting were also at increased risk (HR 1.20 and 1.18, p = 0.006 and p = 0.012, respectively). Among those admitted to the hospital, the risk of subsequent mental health diagnosis increased by 1.5% per day; in the first 31 days of hospitalization, the risk of PTSD diagnosis increased by 13% per day. CONCLUSION: Children who sustain a traumatic injury are at increased risk of developing a mental health condition. PTSD rates found in our real world analysis are lower than those found in prospective studies, raising the possibility of under-recognition of PTSD in this population. LEVEL OF EVIDENCE: Level II.

Mesenchymal stem cells and local tacrolimus delivery synergistically enhance neurite extension.

BACKGROUND: The aim of this study was to investigate the combined effect of mesenchymal stem cells (MSC) and local delivery of tacrolimus (FK506) on nerve regeneration when applied to nerve autografts and decellularized allografts. METHODS: A three-dimensional in vitro compartmented cell culture system consisting of a neonatal dorsal root ganglion adjacent to a nerve graft was used to evaluate the regenerating neurites into the peripheral nerve scaffold. Nerve autografts and allografts were treated with (i) undifferentiated MSCs, (ii) FK506 (100 ng/mL) or (iii) both (N = 9/group). After 48 hours, neurite extension was measured to quantify nerve regeneration and stem cell viability was evaluated. RESULTS: Stem cell viability was confirmed in all MSC-treated grafts. Neurite extension was superior in autografts treated with FK506, and MSCs and FK506 combined (p < 0.001 and p = 0.0001, respectively), and autografts treated with MSCs (p = 0.12) were comparable to untreated autografts. In allografts, FK506 treatment and combined treatment were superior to controls (p < 0.001 and p = 0.0001, respectively), and treatment with MSCs (p = 0.09) was comparable to controls. All autograft groups were superior compared to their respective allograft treatment group (p < 0.05) in neurite extension. CONCLUSIONS: Alone, either MSC or FK506 treatment improved neurite outgrowth, and combined they further enhanced neurite extension in both autografts and allografts.

Multi-scale structuring of cell-instructive cellulose nanocrystal composite hydrogel sheets via sequential electrospinning and thermal wrinkling.

Structured hydrogel sheets offer the potential to mimic the mechanics and morphology of native cell environments in vitro; however, controlling the morphology of such sheets across multiple length scales to give cells consistent multi-dimensional cues remains challenging. Here, we demonstrate a simple two-step process based on sequential electrospinning and thermal wrinkling to create nanocomposite poly(oligoethylene glycol methacrylate)/cellulose nanocrystal hydrogel sheets with a highly tunable multi-scale wrinkled (micro) and fibrous (nano) morphology. By varying the time of electrospinning, rotation speed of the collector, and geometry of the thermal wrinkling process, the hydrogel nanofiber density, fiber alignment, and wrinkle geometry (biaxial or uniaxial) can be independently controlled. Adhered C2C12 mouse myoblast muscle cells display a random orientation on biaxially wrinkled sheets but an extended morphology (directed preferentially along the wrinkles) on uniaxially wrinkled sheets. While the nanofiber orientation had a smaller effect on cell alignment, parallel nanofibers promoted improved cell alignment along the wrinkle direction while perpendicular nanofibers disrupted alignment. The highly tunable structures demonstrated are some of the most complex morphologies engineered into hydrogels to-date without requiring intensive micro/nanofabrication approaches and offer the potential to precisely regulate cell-substrate interactions in a "2.5D" environment (i.e. a surface with both micro- and nano-structured topographies) for in vitro cell screening or in vivo tissue regeneration. STATEMENT OF SIGNIFICANCE: While structured hydrogels can mimic the morphology of natural tissues, controlling this morphology over multiple length scales remains challenging. Furthermore, the incorporation of secondary morphologies within individual hydrogels via simple manufacturing techniques would represent a significant advancement in the field of structured biomaterials and an opportunity to study complex cell-biomaterial interactions. Herein, we leverage a two-step process based on electrospinning and thermal wrinkling to prepare structured hydrogels with microscale wrinkles and nanoscale fibers. Fiber orientation/density and wrinkle geometry can be independently controlled during the electrospinning and thermal wrinkling processes respectively, demonstrating the flexibility of this technique for creating well-defined multiscale hydrogel structures. Finally, we show that while wrinkle geometry is the major determinant of cell alignment, nanofiber orientation also plays a role in this process.

Combined local delivery of tacrolimus and stem cells in hydrogel for enhancing peripheral nerve regeneration.

The application of scaffold-based stem cell transplantation to enhance peripheral nerve regeneration has great potential. Recently, the neuroregenerative potential of tacrolimus (a U.S. Food and Drug Administration-approved immunosuppressant) has been explored. In this study, a fibrin gel-based drug delivery system for sustained and localized tacrolimus release was combined with rat adipose-derived mesenchymal stem cells (MSC) to investigate cell viability in vitro. Tacrolimus was encapsulated in poly(lactic-co-glycolic) acid (PLGA) microspheres and suspended in fibrin hydrogel, using concentrations of 0.01 and 100 ng/ml. Drug release over time was measured. MSCs were cultured in drug-released media collected at various days to mimic systemic exposure. MSCs were combined with (i) hydrogel only, (ii) empty PLGA microspheres in the hydrogel, (iii) 0.01, and (iv) 100 ng/ml of tacrolimus PLGA microspheres in the hydrogel. Stem cell presence and viability were evaluated. A sustained release of 100 ng/ml tacrolimus microspheres was observed for up to 35 days. Stem cell presence was confirmed and cell viability was observed up to 7 days, with no significant differences between groups. This study suggests that combined delivery of 100 ng/ml tacrolimus and MSCs in fibrin hydrogel does not result in cytotoxic effects and could be used to enhance peripheral nerve regeneration.

Local FK506 drug delivery enhances nerve regeneration through fresh, unprocessed peripheral nerve allografts.

OBJECTIVE: Nerve allografts offer many advantages in the reconstruction of peripheral nerve gaps: they retain their native microstructure, contain pro-regenerative Schwann cells, are widely available, and avoid donor site morbidity. Unfortunately, clinical use of nerve allografts is limited by the need for systemic immunosuppression and its adverse effects. To eliminate the toxicity of the systemic immunosuppressant FK506, we developed a local FK506 drug delivery system (DDS) to provide drug release over 28 days. The study objective was to investigate if the local FK506 DDS enhances nerve regeneration in a rodent model of nerve gap defect reconstruction with immunologically-disparate nerve allografts. METHODS: In male Lewis rats, a common peroneal nerve gap defect was reconstructed with either a 20 mm nerve isograft from a donor Lewis rat or a 20 mm fresh, unprocessed nerve allograft from an immunologically incompatible donor ACI rat. After 4 weeks of survival, nerve regeneration was evaluated using retrograde neuronal labelling, quantitative histomorphometry, and serum cytokine profile. RESULTS: Treatment with both systemic FK506 and the local FK506 DDS significantly improved motor and sensory neuronal regeneration, as well as histomorphometric indices including myelinated axon number. Rats with nerve allografts treated with either systemic or local FK506 had significantly reduced serum concentrations of the pro-inflammatory cytokine IL-12 compared to untreated vehicle control rats with nerve allografts. Serum FK506 levels were undetectable in rats with local FK506 DDS. INTERPRETATION: The local FK506 DDS improved motor and sensory nerve regeneration through fresh nerve allografts to a level equal to that of either systemic FK506 or nerve isografting. This treatment may be clinically translatable in peripheral nerve reconstruction or vascularized composite allotransplantation.

The obstetrical research landscape: a cross-sectional analysis of clinical trials from 2007-2020.

Background: Obstetrical complications affect more than a third of women globally, but are underrepresented in clinical research. Little is known about the comprehensive obstetrical clinical trial landscape, how it compares with other fields, or factors associated with the successful completion of obstetrical trials. Objective: This study aimed to characterize obstetrical clinical trials registered on ClinicalTrials.gov with the primary objective of identifying features associated with early discontinuation and results reporting. Study Design: This is a cross-sectional study with descriptive, logistic regression and Cox regression analyses of clinical trials registered on ClinicalTrials.gov. Our primary exposure variables were trial focus (obstetrical or nonobstetrical) and trial funding (industry, United States government, or academic). We conducted additional exploratory analyses of other trial features including design, enrollment, and therapeutic focus. We examined the associations of exposure variables and other trial features with 2 primary outcomes: early discontinuation and results reporting. Results: We downloaded data for all studies (N=332,417) registered on ClinicalTrials.gov from October 1, 2007, to March 9, 2020, from the Aggregate Analysis of ClinicalTrials.gov database. We excluded studies with a noninterventional design (n=63,697) and those registered before October 1, 2007 (n=45,209). A total of 4276 obstetrical trials (1.9%) (ie, interventional studies) and 219,235 nonobstetric trials (98.1%) were compared. Among all trials, 2.8% of academic-funded trials, 1.9% of United States government-funded trials, and 0.4% of industry-funded trials focused on obstetrics. The quantity of obstetrical trials increased over time (10.8% annual growth rate). Compared with nonobstetrical trials, obstetrical trials had a greater risk of early discontinuation (adjusted hazard ratio, 1.40; 95% confidence interval, 1.21-1.62; P<.0001) and similar odds of results reporting (adjusted odds ratio, 0.89; 95% confidence interval, 0.72-1.10; P=.19). Among obstetrical trials funders after controlling for confounding variables, United States government-funded trials were at the lowest risk of early discontinuation (United States government, adjusted hazard ratio, 0.23; 95% confidence interval, 0.07-0.69; P=.009; industry reference; academic, adjusted hazard ratio, 1.04; 95% confidence interval, 0.62-1.74; P=.88). Academic-funded trials had the lowest odds of results reporting after controlling for confounding variables (academic institutions, adjusted odds ratio, 0.39; 95% confidence interval, 0.22-0.68; P=.0009; industry reference; United States government, adjusted odds ratio, 1.06; 95% confidence interval, 0.53-2.09; P=.87). Conclusion: Obstetrical trials represent only 1.9% of all clinical trials in ClinicalTrials.gov and have comparatively poor completion. All stakeholders should commit to increasing the number of obstetrical trials and improving their completion and dissemination to ensure clinical research reflects the obstetrical burden of disease and advances maternal health.

Désinfection à la chaleur humide des respirateurs N95, inactivation du SRAS-CoV-2 et effets sur les propriétés des respirateurs.

CONTEXTE: La demande sans précédent de respirateurs N95 durant la pandémie de maladie à coronavirus 2019 (COVID-19) a entraîné une pénurie mondiale. Nous avons validé un protocole de décontamination rapide et économique répondant aux normes réglementaires afin de permettre la réutilisation sûre de ce type de masque. MÉTHODES: Nous avons contaminé 4 modèles courants de respirateurs N95 avec le coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) et avons évalué l'inactivation virale après une désinfection de 60 minutes à 70 °C et à une humidité relative de 0 %. De même, nous avons étudié l'efficacité de la désinfection thermique, à une humidité relative allant de 0 % à 70 %, de masques contaminés à Escherichia coli. Enfin, nous avons examiné des masques soumis à de multiples cycles de désinfection thermique: nous avons évalué leur intégrité structurelle à l'aide d'un microscope à balayage, et leurs propriétés protectrices au moyen des normes du National Institute for Occupational Safety and Health des États-Unis relatives à la filtration particulaire, à la résistance respiratoire et à l'ajustement. RÉSULTATS: Une seule désinfection thermique a suffi pour que le SRAS-CoV-2 ne soit plus décelable sur les masques étudiés. En ce qui concerne les masques contaminés à E. coli, une culture de 24 heures a révélé que la bactérie n'était pratiquement plus décelable sur les masques désinfectés à 70 °C et à une humidité relative de 50 %, contrairement aux masques non désinfectés (densité optique à une longueur d'onde de 600 nm : 0,02 ± 0,02 contre 2,77 ± 0,09; p < 0,001), mais qu'elle persistait sur les masques traités à une humidité relative moindre. Les masques ayant subi 10 cycles de désinfection avaient toujours des fibres de diamètre semblable à celui des fibres des masques non traités, et ils répondaient encore aux normes d'ajustement, de filtration et de résistance respiratoire. INTERPRÉTATION: La désinfection thermique a réussi à décontaminer les respirateurs N95 sans compromettre leur intégrité structurelle ni modifier leurs propriétés. Elle pourrait se faire dans les hôpitaux et les établissements de soins de longue durée avec de l'équipement facilement accessible, ce qui réduirait la pénurie de N95.

Effect of moist heat reprocessing of N95 respirators on SARS-CoV-2 inactivation and respirator function.

BACKGROUND: Unprecedented demand for N95 respirators during the coronavirus disease 2019 (COVID-19) pandemic has led to a global shortage of these masks. We validated a rapidly applicable, low-cost decontamination protocol in compliance with regulatory standards to enable the safe reuse of N95 respirators. METHODS: We inoculated 4 common models of N95 respirators with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and evaluated viral inactivation after disinfection for 60 minutes at 70°C and 0% relative humidity. Similarly, we evaluated thermal disinfection at 0% to 70% relative humidity for masks inoculated with Escherichia coli. We assessed masks subjected to multiple cycles of thermal disinfection for structural integrity using scanning electron microscopy and for protective functions using standards of the United States National Institute for Occupational Safety and Health for particle filtration efficiency, breathing resistance and respirator fit. RESULTS: A single heat treatment rendered SARS-CoV-2 undetectable in all mask samples. Compared with untreated inoculated control masks, E. coli cultures at 24 hours were virtually undetectable from masks treated at 70°C and 50% relative humidity (optical density at 600 nm wavelength, 0.02 ± 0.02 v. 2.77 ± 0.09, p < 0.001), but contamination persisted for masks treated at lower relative humidity. After 10 disinfection cycles, masks maintained fibre diameters similar to untreated masks and continued to meet standards for fit, filtration efficiency and breathing resistance. INTERPRETATION: Thermal disinfection successfully decontaminated N95 respirators without impairing structural integrity or function. This process could be used in hospitals and long-term care facilities with commonly available equipment to mitigate the depletion of N95 masks.

Systemic and Local FK506 (Tacrolimus) and its Application in Peripheral Nerve Surgery.

Peripheral nerve injuries (PNI) are common and frequently afflict otherwise healthy individuals after traumatic or iatrogenic events. Adjuvant therapies to improve functional outcomes after surgical repair of PNI have been investigated extensively in preclinical studies; however, to date, none have been clinically proven to have a notable therapeutic effect. FK506 (tacrolimus), a US Food and Drug Administration-approved systemic immunosuppressant, has demonstrated promising neuro-regenerative properties in both animal studies and clinical reports, but its adverse effects when systemically administered have precluded its broader applicability for patients with PNI. Recent advances in bioengineered drug delivery systems have made local FK506 delivery to a site of PNI an intriguing method of promoting peripheral nerve regeneration, with promising results in preclinical translational investigations. This review summarizes the preclinical and clinical evidence for FK506's beneficial effect in promoting peripheral nerve regeneration when administered systemically and locally.

A single session of brief electrical stimulation enhances axon regeneration through nerve autografts.

Nerve graft reconstruction of gap defects may result in poor clinical outcomes, particularly with long regeneration distances. Electrical stimulation (ES) of nerves may improve outcomes in such patients. A single session of ES at 20 Hz for 1 h significantly enhances axon regeneration in animals and human subjects after nerve crush or nerve transection and repair. The objectives of this study were to evaluate if ES enhances axon regeneration through nerve grafts and if there is added benefit of a second, delayed session of ES (serial ES) on axon regeneration as compared to a single session only of ES. In female rats, a gap defect was created in the hindlimb common peroneal (CP) nerve and immediately reconstructed with a 10 mm nerve autograft (Experiment 1) or a 20 mm nerve autograft (Experiment 2). In Experiment 1, rats were randomized to 1 h of CP nerve ES or sham stimulation. In Experiment 2, rats were randomized to control (sham ES + sham ES), single ES (ES + sham ES), or serial ES (ES + ES), which consisted of an initial 1 h session of either ES or sham stimulation of the CP nerve, followed by a second 1 h session of ES or sham stimulation of the CP nerve 4 weeks later. In both experiments, after a 6 week period of nerve regeneration, CP neurons that had regenerated axons distal to the autograft were retrograde labelled for enumeration, and the CP nerve distal to the autograft was harvested for histomorphometry. In Experiment 1, rats that received CP nerve ES had statistically significantly more motor (p < .05) and sensory (p < .05) neurons that regenerated axons distal to the 10 mm nerve autograft, with more myelinated axons on histomorphometry (p < .001). Similarly, in Experiment 2, significantly more motor (p < .01) and sensory (p < .05) neurons regenerated axons distal to the 20 mm nerve autograft after a single session or two sessions of CP nerve ES. There was no significant difference in the number of regenerated motor or sensory neurons between rats with 20 mm CP nerve autografts receiving either one or two sessions of CP nerve ES (p > .05). In conclusion, a single session of ES enhances axon regeneration following nerve autografting with no added effect of a second, delayed session of ES. These findings support previous studies in animals and humans of the robust effect of a single session of ES in promoting nerve regeneration following injury and repair.

Local delivery of FK506 to injured peripheral nerve enhances axon regeneration after surgical nerve repair in rats.

Administration of FK506, an FDA approved immunosuppressant, has been shown to enhance nerve regeneration following peripheral nerve injuries. However, the severe side effects of the systemically delivered FK506 has prevented clinicians from the routine use of the drug. In this study, we analyzed the effectiveness of our fibrin gel-based FK506 delivery system to promote axon regeneration in a rat peripheral nerve transection and immediate surgical repair model. In addition, biodistribution of FK506 from the local delivery system to the surrounding tissues was analyzed in vivo. Rats in the negative control groups either did not receive any delivery system treatment or received fibrin gel with empty microspheres. The experimental groups included rats treated with fibrin gel loaded with solubilized, particulate, and poly(lactic-co-glycolic) acid microspheres-encapsulated FK506. Rats in experimental groups receiving FK506 microspheres and the particulate FK506 regenerated the highest number of motor and sensory neurons. Histomorphometric analysis also demonstrated greater numbers of myelinated axons following particulate FK506 and FK506 microspheres treatment compared to the negative control groups. In biodistribution studies, FK506 was found at the nerve repair site, the sciatic nerve, and spinal cord, with little to no drug detection in other vital organs. Hence, the local application of FK506 via our delivery systems enhanced axon regeneration whilst avoiding the toxicity of systemic FK506. This local delivery strategy represents a new opportunity for clinicians to use for cases of peripheral nerve injuries. STATEMENT OF SIGNIFICANCE: This work for the first time investigated the influence of locally administered FK506 to the site of nerve injury and immediate repair directly on the number of motor and sensory neurons that regenerated their axons. Furthermore, using the immediate nerve repair model, we obtained valuable information about the biodistribution of FK506 within the nervous system following its release from the delivery system implanted at the site of nerve injury and repair. The strategy of local FK506 delivery holds a great promise in the clinical translation, as the localized delivery circumvents the main limitation of the systemic delivery of FK506, that of immunosuppression and toxicity.

Local FK506 dose-dependent study using a novel three-dimensional organotypic assay.

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a three-dimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG-ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone.

Matrices, scaffolds, and carriers for protein and molecule delivery in peripheral nerve regeneration.

Local application of exogenous agents with neurotrophic properties enhances the regenerative capacity of injured neurons, especially following reconstructions of long nerve gaps and delayed nerve repairs. Recent advances in biomaterials and biomedical engineering have provided options for the sustained and controlled release of macromolecules to the peripheral nerve. Here, we review five methods for delivering macromolecules to the peripheral nerve including mini-osmotic pumps, hydrogel-based delivery systems, nerve guidance conduits, electrospun fibers, and nerve wraps. In addition to controlling the release of bioactive macromolecules, the ease of clinical use and versatility in implantation at a variety of "real-world" anatomical locations are key factors in designing an ideal delivery system. The incorporation of both mechanical and biological cues into such devices also helps optimize these systems.

Injectable Anisotropic Nanocomposite Hydrogels Direct in Situ Growth and Alignment of Myotubes.

While injectable in situ cross-linking hydrogels have attracted increasing attention as minimally invasive tissue scaffolds and controlled delivery systems, their inherently disorganized and isotropic network structure limits their utility in engineering oriented biological tissues. Traditional methods to prepare anisotropic hydrogels are not easily translatable to injectable systems given the need for external equipment to direct anisotropic gel fabrication and/or the required use of temperatures or solvents incompatible with biological systems. Herein, we report a new class of injectable nanocomposite hydrogels based on hydrazone cross-linked poly(oligoethylene glycol methacrylate) and magnetically aligned cellulose nanocrystals (CNCs) capable of encapsulating skeletal muscle myoblasts and promoting their differentiation into highly oriented myotubes in situ. CNC alignment occurs on the same time scale as network gelation and remains fixed after the removal of the magnetic field, enabling concurrent CNC orientation and hydrogel injection. The aligned hydrogels show mechanical and swelling profiles that can be rationally modulated by the degree of CNC alignment and can direct myotube alignment both in two- and three-dimensions following coinjection of the myoblasts with the gel precursor components. As such, these hydrogels represent a critical advancement in anisotropic biomimetic scaffolds that can be generated noninvasively in vivo following simple injection.

µFE models can represent microdamaged regions of healthy and metastatically involved whole vertebrae identified through histology and contrast enhanced µCT imaging.

Micro-damage formation within the skeleton is an important stimulant for bone remodeling, however abnormal build-up of micro-damage can lead to skeletal fragility. In this study, µCT imaging based micro finite element (µFE) models were used to evaluate tissue level damage criteria in whole healthy and metastatically-involved vertebrae. T13-L2 spinal segments were excised from osteolytic (n=3) and healthy (n=3) female athymic rnu/rnu rats. Osteolytic metastasis was generated by intercardiac injection of HeLa cancer cells. Micro-mechanical axial loading was applied to the spinal motion segments under µCT imaging. Vertebral samples underwent BaSO4 staining and sequential calcein/fuchsin staining to identify load induced micro-damage. µCT imaging was used generate specimen specific µFE models of the healthy and osteolytic whole rat vertebrae. Model boundary conditions were generated through deformable image registration of loaded and unloaded scans. Elevated stresses and strains were detected in regions of micro-damage identified through histological and BaSO4 staining within healthy and osteolytic vertebral models, as compared to undamaged regions. Additionally, damaged regions of metastatic vertebrae experienced significantly higher local stresses and strains than those in the damaged regions of healthy specimens. Areas identified by BaSO4 staining, however, yielded lower levels of stress and strain in damaged and undamaged regions of healthy and metastatic vertebrae as compared to fuschin staining. The multimodal (experimental, image-based and computational) techniques used in this study demonstrated the ability of local stresses and strains computed through µFE analysis to identify trabecular micro-damage, that can be applied to biomechanical analyses of healthy and diseased whole bones.

Enhanced Mechanical Properties in Cellulose Nanocrystal-Poly(oligoethylene glycol methacrylate) Injectable Nanocomposite Hydrogels through Control of Physical and Chemical Cross-Linking.

While injectable hydrogels have several advantages in the context of biomedical use, their generally weak mechanical properties often limit their applications. Herein, we describe in situ-gelling nanocomposite hydrogels based on poly(oligoethylene glycol methacrylate) (POEGMA) and rigid rod-like cellulose nanocrystals (CNCs) that can overcome this challenge. By physically incorporating CNCs into hydrazone cross-linked POEGMA hydrogels, macroscopic properties including gelation rate, swelling kinetics, mechanical properties, and hydrogel stability can be readily tailored. Strong adsorption of aldehyde- and hydrazide-modified POEGMA precursor polymers onto the surface of CNCs promotes uniform dispersion of CNCs within the hydrogel, imparts physical cross-links throughout the network, and significantly improves mechanical strength overall, as demonstrated by quartz crystal microbalance gravimetry and rheometry. When POEGMA hydrogels containing mixtures of long and short ethylene oxide side chain precursor polymers were prepared, transmission electron microscopy reveals that phase segregation occurs with CNCs hypothesized to preferentially locate within the stronger adsorbing short side chain polymer domains. Incorporating as little as 5 wt % CNCs results in dramatic enhancements in mechanical properties (up to 35-fold increases in storage modulus) coupled with faster gelation rates, decreased swelling ratios, and increased stability versus hydrolysis. Furthermore, cell viability can be maintained within 3D culture using these hydrogels independent of the CNC content. These properties collectively make POEGMA-CNC nanocomposite hydrogels of potential interest for various biomedical applications including tissue engineering scaffolds for stiffer tissues or platforms for cell growth.