MicroRNA-based therapeutics for inflammatory disorders of the microbiota-gut-brain axis.

An emerging but less explored shared pathophysiology across microbiota-gut-brain axis disorders is aberrant miRNA expression, which may represent novel therapeutic targets. miRNAs are small, endogenous non-coding RNAs that are important transcriptional repressors of gene expression. Most importantly, they regulate the integrity of the intestinal epithelial and blood-brain barriers and serve as an important communication channel between the gut microbiome and the host. A well-defined understanding of the mode of action, therapeutic strategies and delivery mechanisms of miRNAs is pivotal in translating the clinical applications of miRNA-based therapeutics. Accumulating evidence links disorders of the microbiota-gut-brain axis with a compromised gut-blood-brain-barrier, causing gut contents such as immune cells and microbiota to enter the bloodstream leading to low-grade systemic inflammation. This has the potential to affect all organs, including the brain, causing central inflammation and the development of neurodegenerative and neuropsychiatric diseases. In this review, we have examined in detail miRNA biogenesis, strategies for therapeutic application, delivery mechanisms, as well as their pathophysiology and clinical applications in inflammatory gut-brain disorders. The research data in this review was drawn from the following databases: PubMed, Google Scholar, and Clinicaltrials.gov. With increasing evidence of the pathophysiological importance for miRNAs in microbiota-gut-brain axis disorders, therapeutic targeting of cross-regulated miRNAs in these disorders displays potentially transformative and translational potential. Further preclinical research and human clinical trials are required to further advance this area of research.

Intraoperative Image-Guided Navigation in Craniofacial Surgery: Review and Grading of the Current Literature.

INTRODUCTION: Image-guided navigation has existed for nearly 3 decades, but its adoption to craniofacial surgery has been slow. A systematic review of the literature was performed to assess the current status of navigation in craniofacial surgery. METHODS: A Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) systematic review of the Medline and Web of Science databases was performed using a series of search terms related to Image-Guided Navigation and Craniofacial Surgery. Titles were then filtered for relevance and abstracts were reviewed for content. Single case reports were excluded as were animal, cadaver, and virtual data. Studies were categorized based on the type of study performed and graded using the Jadad scale and the Newcastle-Ottawa scales, when appropriate. RESULTS: A total of 2030 titles were returned by our search criteria. Of these, 518 abstracts were reviewed, 208 full papers were evaluated, and 104 manuscripts were ultimately included in the study. A single randomized controlled trial was identified (Jadad score 3), and 12 studies were identified as being case control or case cohort studies (Average Newcastle-Ottawa score 6.8) The most common application of intraoperative surgical navigation cited was orbital surgery (n = 36), followed by maxillary surgery (n = 19). Higher quality studies more commonly pertained to the orbit (6/13), and consistently show improved results. CONCLUSION: Image guided surgical navigation improves outcomes in orbital reconstruction. Although image guided navigation has promise in many aspects of craniofacial surgery, current literature is lacking and future studies addressing this paucity of data are needed before universal adoption can be recommended.

Prolonged Cold Ischemia Time Results in Local and Remote Organ Dysfunction in a Murine Model of Vascularized Composite Transplantation.

Vascularized composite allotransplantation (VCA) is a viable reconstructive option for complex tissue defects. Although grafts with a large muscular component may be uniquely susceptible to ischemia-reperfusion (I/R) syndrome, the safe cold ischemia time in VCA has not been established. We investigated the effects of cold ischemia on innate immune response and recipient survival in a murine orthotopic hindlimb transplantation model. Surprisingly, mice receiving grafts exposed to 6 h or longer of cold storage demonstrated reduced survival and massive elevations in serum creatinine, blood urea nitrogen, and creatine kinase, compared with 1 h of cold storage recipients. This was accompanied by progressive increase in macrophage and neutrophil cell infiltration in muscle biopsy specimens, altered platelet endothelial cell adhesion molecule-1 expression, and ultimate renal injury. Multiplex immunoassay analysis identified 21 cytokines in serum and 18 cytokines in muscle biopsy specimens that are likely essential in the complex response to I/R-triggered injury in VCA. In conclusion, this study, in a mouse model of orthotopic hindlimb transplantation, is the first to document that prolonged cold ischemia triggers progressive I/R injury with vascular endothelial damage and may lead to irrecoverable local and remote organ damage. These experimental findings are important in guiding future therapies for human VCA recipients.

Procurement strategies for combined multiorgan and composite tissues for transplantation.

PURPOSE OF REVIEW: The purpose of this article is to identify the unique aspects of combined multiorgan and vascularized composite allograft (VCA) procurement from deceased donors and outline the steps essential for success. RECENT FINDINGS: Transplantation of nonsolid organ composite tissues is becoming a viable option for reconstruction of massive tissue defects. With the United Network for Organ Sharing designation of VCAs as organs, placing them under the domain of the Organ Procurement and Transplantation Network, a systematic method for combined solid organ and VCA procurement is required. Several centers have reported experience with successful procurement strategies including sequential and simultaneous retrievals. The published literature describing donor screening, sequence of procurement with relation to solid organs and allocation is reviewed. SUMMARY: With the 2013 classification of VCAs as organs, the Organ Procurement and Transplantation Network and United Network for Organ Sharing are better suited to aligning procurement and allocation policies. As VCA transplantation becomes more commonplace, protocol guidelines will ensure smooth integration with existing procurement infrastructure.

Case Series: Efficacy of Polyclonal Intravenous Immunoglobulin for Refractory Clostridioides difficile Infection.

BACKGROUND: Intravenous immunoglobulin (IVIg) for Clostridioides difficile infection (CDI) no longer features in treatment guidelines. However, IVIg is still used by some clinicians for severe or recurrent CDI (rCDI) cases. The main objective of this study was to investigate the efficacy of IVIg and to identify possible predictors of disease resolution post IVIg administration for patients with CDI. METHODS: This retrospective observational cohort study of patients ≥2 years old hospitalised with severe, relapsing, or rCDI treated with IVIg therapy was performed in a large UK tertiary hospital between April 2018 and March 2023. Scanned electronic notes from patient admissions and clinical reporting systems were used to collect relevant data. RESULTS: In total, 20/978 patients diagnosed with CDI over the 5-year study were treated with IVIg. Twelve (60%) had hospital-onset CDI. Eleven of the twenty patients (55%) responded to treatment, with a mean of 8.6 (SD 10.7) days to disease resolution. Sixteen (80%) patients were treated for severe CDI and four (20%) for rCDI (n = 3) and relapsing CDI (n = 1). There were no statistically significant differences in possible independent predictors of disease resolution post IVIg administration between groups. There was an average of 6.2 (4.9) days to IVIg administration after diagnosis with no difference between responders and non-responders (p = 0.88) and no further significant difference in additional indicators. Four (36%) of the responders were immunosuppressed compared to just one (11%) of the non-responders (p = 0.15). Six of the responders (two with recurrent and four with severe CDI) improved rapidly within 2 days, and three of these were immunosuppressed. CONCLUSION: We observed disease resolution post IVIg therapy in over 50% of patients with refractory CDI. Our data also support a potential enhanced effect of IVIg in immunosuppressed individuals. Thus, the role of IVIg for CDI treatment, particularly in the immunosuppressed, warrants future case-control studies coupled to mechanistic investigations to improve care for this ongoing significant healthcare-associated infection.

Reducing Risks for Midface and Mandible Fracture Repair.

The midface skeleton provides structural scaffolding to the middle third of the face. Complications associated with fracture repair in these regions can result from incomplete, inaccurate, or delayed assessment, poor initial and subsequent reduction and fixation, infection, uncontrolled hemorrhage, hardware failure and associated soft tissue injuries. A systematic approach to managing the patient with facial trauma that includes Acute Trauma Life Support principles, early reconstruction, and precise reduction and fixation is essential to reducing the short-term and long-term risks of complications.

A photoactivated nanofiber graft material for augmented Achilles tendon repair.

BACKGROUND AND OBJECTIVE: Suture repair of Achilles tendon rupture can cause infection, inflammation and scarring, while prolonged immobilization promotes adhesions to surrounding tissues and joint stiffness. Early mobilization can reduce complications provided the repair is strong enough to resist re-rupture. We have developed a biocompatible, photoactivated tendon wrap from electrospun silk (ES) to provide additional strength to the repair that could permit early mobilization, and act as a barrier to adhesion formation. STUDY DESIGN/MATERIAL AND METHODS: ES nanofiber mats were prepared by electrospinning. New Zealand white rabbits underwent surgical transection of the Achilles tendon and repair by: (a) SR: standard Kessler suture + epitendinous suture (5-0 vicryl). (b) ES/PTB: a single stay suture and a section of ES mat, stained with 0.1% Rose Bengal (RB), wrapped around the tendon and bonded with 532 nm light (0.3 W/cm(2) , 125 J/cm(2) ). (c) SR + ES/PTB: a combination of (a) and (b). Gross appearance, extent of adhesion formation and biomechanical properties of the repaired tendon were evaluated at Days 7, 14, or 28 post-operatively (n = 8 per group at each time point). RESULTS: Ultimate stress (US) and Young's modulus (E) in the SR group were not significantly different from the ES/PTB group at Days 7 (US, P = 0.85; E, P = 1), 14 (US, P = 0.054; E, P = 1), and 28 (US, P = 0.198; E, P = 0.12) post-operatively. Adhesions were considerably greater in the SR group compared to the ES/PTB group at Days 7 (P = 0.002), 14 (P < 0.0001), and 28 (P < 0.0001). The combination approach of SR + ES/PTB gave the best outcomes in terms of E at 7 (P < 0.016) and 14 days (P < 0.016) and reduced adhesions compared to SR at 7 (P < 0.0001) and 14 days (P < 0.0001), the latter suggesting a barrier function for the photobonded ES wrap. CONCLUSION: Photochemical sealing of a ES mat around the tendon repair site provides considerable benefit in Achilles tendon repair. Lasers Surg. Med. 44: 645-652, 2012. © 2012 Wiley Periodicals, Inc.

Centralized, capacity-building training of Lichtenstein hernioplasty in Brazil.

BACKGROUND: In Brazil, access to healthcare varies widely by community. Options for repair of surgically correctable conditions, such as inguinal hernias, are limited. A training program was instituted to expand access to Lichtenstein hernioplasty. METHODS: Between September, 2014 and September, 2015, 3 orders of training series took place in São Paulo, Brazil. Participating surgeons received training and assessments from expert trainers using the Operative Performance Rating Scale (OPRS). Those who completed training successfully were invited to become trainers. OPRS scores were compared between training series. Outcomes were documented up to 6 months post-training. RESULTS: The 3 orders of training series resulted in 45 surgeons trained and 213 hernias repaired. Eleven trainees subsequently became trainers. Mean post-training OPRS scores were 4.4 (scale of 5) and did not vary significantly between training series. The overall complication rate was 4.7%, with no hernia recurrences or reoperations at 6 months. CONCLUSIONS: Competency-based training generates a regional network of surgeons proficient in Lichtenstein hernioplasty. Each training session progressively expands patient access to high quality operations in underserved communities in Brazil.

Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells.

Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze-thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5 x 10(5) cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3 x 10(6) cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (+/- 13.6) microg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.

Scaffold mesh size affects the osteoblastic differentiation of seeded marrow stromal cells cultured in a flow perfusion bioreactor.

In this study, we cultured marrow stromal cells on titanium fiber meshes in a flow perfusion bioreactor and examined the effect of altering scaffold mesh size on cell behavior in an effort to develop a bone tissue construct composed of a scaffold, osteogenic cells, and extracellular matrix. Scaffolds of differing mesh size, that is, distance between fibers, were created by altering the diameter of the mesh fibers (20 or 40 microm) while maintaining a constant porosity. These scaffolds had a porosity of 80% and mesh sizes of 65 microm (20-microm fibers) or 119 microm (40-microm fibers). Cell/scaffold constructs were grown in static culture or under flow for up to 16 days and assayed for osteoblastic differentiation. Cellularity was higher at early time points and Ca2+ deposition was higher at later time points for flow constructs over static controls. The 20-microm mesh had reduced cellularity in static culture. Under flow conditions, mass transport limitations are mitigated allowing uniform cell growth throughout the scaffold, and there was no difference in cellularity between mesh types. There was greater alkaline phosphatase (ALP) activity, osteopontin levels, and calcium under flow at 8 days for the 40-microm mesh compared to the 20-microm mesh. However, by day 16, the trend was reversed, suggesting the time course of differentiation was dependent on scaffold mesh size under flow conditions. However, this dependence was not linear with respect to time; larger mesh size was conducive to early osteoblast differentiation while smaller mesh size was conducive to later differentiation and matrix deposition.

Wearable Technology for Global Surgical Teleproctoring.

OBJECTIVE: In underserved communities around the world, inguinal hernias represent a significant burden of surgically-treatable disease. With traditional models of international surgical assistance limited to mission trips, a standardized framework to strengthen local healthcare systems is lacking. We established a surgical education model using web-based tools and wearable technology to allow for long-term proctoring and assessment in a resource-poor setting. This is a feasibility study examining wearable technology and web-based performance rating tools for long-term proctoring in an international setting. METHODS: Using the Lichtenstein inguinal hernia repair as the index surgical procedure, local surgeons in Paraguay and Brazil were trained in person by visiting international expert trainers using a formal, standardized teaching protocol. Surgeries were captured in real-time using Google Glass and transmitted wirelessly to an online video stream, permitting real-time observation and proctoring by mentoring surgeon experts in remote locations around the world. A system for ongoing remote evaluation and support by experienced surgeons was established using the Lichtenstein-specific Operative Performance Rating Scale. RESULTS: Data were collected from 4 sequential training operations for surgeons trained in both Paraguay and Brazil. With continuous internet connectivity, live streaming of the surgeries was successful. The Operative Performance Rating Scale was immediately used after each operation. Both surgeons demonstrated proficiency at the completion of the fourth case. CONCLUSIONS: A sustainable model for surgical training and proctoring to empower local surgeons in resource-poor locations and "train trainers" is feasible with wearable technology and web-based communication. Capacity building by maximizing use of local resources and expertise offers a long-term solution to reducing the global burden of surgically-treatable disease.

Effect of fiber diameter, pore size and seeding method on growth of human dermal fibroblasts in electrospun poly(epsilon-caprolactone) fibrous mats.

Nonwoven fiber mats of poly(epsilon-caprolactone) (PCL) and PCL blended with poly(ethylene oxide) (PEO) were generated by electrospinning. Differential scanning calorimetry, scanning electron microscopy, and gravimetric measurement confirm the removal of PEO after immersion in water, as well as an increase in the PCL crystallinity. The reorganization of PCL resulted in the macroscopic alteration of the electrospun mat, decreasing the peak pore diameter up to a factor of 3 while only minimally affecting the fiber diameter. This technique was used to create electrospun PCL scaffolds with similar fiber diameters but different pore diameters to examine the effect of pore diameter on cell growth. Human Dermal Fibroblasts (HDF) were seeded into multiple samples using a perfusion seeding technique to guarantee successful cell deposition. Fluorescence analysis at 7, 14, and 21 days found that cells proliferated at a faster rate on scaffolds with peak pore diameters greater than 6 microm, as determined by mercury porosimetry. Cell conformation was also found to change as the peak pore diameter grew from 12 to 23 microm; cells began aligning along single fibers instead of attaching to multiple fibers. Knowledge of the effect of void architecture on cell proliferation and conformation could lead to the development of more effective scaffolds for tissue engineering.

In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation.

This study instituted a unique approach to bone tissue engineering by combining effects of mechanical stimulation in the form of fluid shear stresses and the presence of bone-like extracellular matrix (ECM) on osteodifferentiation. Rat marrow stromal cells (MSCs) harvested from bone marrow were cultured on titanium (Ti) fiber mesh discs for 12 days in a flow perfusion system to generate constructs containing bone-like ECM. To observe osteodifferentiation and bone-like matrix deposition, these decellularized constructs and plain Ti fiber meshes were seeded with MSCs (Ti/ECM and Ti, respectively) and cultured in the presence of fluid shear stresses either with or without the osteogenic culture supplement dexamethasone. The calcium content, alkaline phosphatase activity, and osteopontin secretion were monitored as indicators of MSC differentiation. Ti/ECM constructs demonstrated a 75-fold increase in calcium content compared with their Ti counterparts after 16 days of culture. After 16 days, the presence of dexamethasone enhanced the effects of fluid shear stress and the bone-like ECM by increasing mineralization 50-fold for Ti/ECM constructs; even in the absence of dexamethasone, the Ti/ECM constructs exhibited approximately a 40-fold increase in mineralization compared with their Ti counterparts. Additionally, denatured Ti/ECM* constructs demonstrated a 60-fold decrease in calcium content compared with Ti/ECM constructs after 4 days of culture. These results indicate that the inherent osteoinductive potential of bone-like ECM along with fluid shear stresses synergistically enhance the osteodifferentiation of MSCs with profound implications on bone-tissue-engineering applications.