3D stem-like spheroids-on-a-chip for personalized combinatorial drug testing in oral cancer.

BACKGROUND: Functional drug testing (FDT) with patient-derived tumor cells in microfluidic devices is gaining popularity. However, the majority of previously reported microfluidic devices for FDT were limited by at least one of these factors: lengthy fabrication procedures, absence of tumor progenitor cells, lack of clinical correlation, and mono-drug therapy testing. Furthermore, personalized microfluidic models based on spheroids derived from oral cancer patients remain to be thoroughly validated. Overcoming the limitations, we develop 3D printed mold-based, dynamic, and personalized oral stem-like spheroids-on-a-chip, featuring unique serpentine loops and flat-bottom microwells arrangement. RESULTS: This unique arrangement enables the screening of seven combinations of three drugs on chemoresistive cancer stem-like cells. Oral cancer patients-derived stem-like spheroids (CD 44+) remains highly viable (> 90%) for 5 days. Treatment with a well-known oral cancer chemotherapy regimen (paclitaxel, 5 fluorouracil, and cisplatin) at clinically relevant dosages results in heterogeneous drug responses in spheroids. These spheroids are derived from three oral cancer patients, each diagnosed with either well-differentiated or moderately-differentiated squamous cell carcinoma. Oral spheroids exhibit dissimilar morphology, size, and oral tumor-relevant oxygen levels (< 5% O2). These features correlate with the drug responses and clinical diagnosis from each patient's histopathological report. CONCLUSIONS: Overall, we demonstrate the influence of tumor differentiation status on treatment responses, which has been rarely carried out in the previous reports. To the best of our knowledge, this is the first report demonstrating extensive work on development of microfluidic based oral cancer spheroid model for personalized combinatorial drug screening. Furthermore, the obtained clinical correlation of drug screening data represents a significant advancement over previously reported personalized spheroid-based microfluidic devices. Finally, the maintenance of patient-derived spheroids with high viability under oral cancer relevant oxygen levels of less than 5% O2 is a more realistic representation of solid tumor microenvironment in our developed device.

Sensory and pain outcomes of neurotized skin-grafted free gracilis muscle flaps for lower extremity reconstruction.

BACKGROUND: Skin-grafted free gracilis muscle flaps are commonly used for lower extremity reconstruction. However, the loss of sensory function may lead to increased patient morbidity. This study prospectively analyzed the sensory and neuropathic pain outcomes of neurotized skin-grafted free gracilis muscle flaps used for the reconstruction of lower extremity defects. METHODS: Patients undergoing lower extremity reconstructions between 2020 and 2022 with neurotized skin-grafted free gracilis muscle flaps were prospectively enrolled. Sensation was assessed at 3, 6 and 12 months postoperatively using monofilaments, two-point discrimination, a vibration device, and cold and warm metal rods. Sensations were tested in the center and periphery of the flaps, as well as in the surrounding skin. The contralateral side served as the control. Patients completed the McGill pain questionnaire to evaluate patient-reported neuropathic pain. RESULTS: Ten patients were included. At 12 months postoperatively, monofilament values improved by 44.5% compared to that of the control site, two-point discrimination, cold detection, warmth detection, and vibration detection improved by 36.2%, 48%, 50%, and 88.2%, respectively, at the reconstructed site compared to those at the control site. All sensory tests were significantly better than 3 and 6 months values (p < 0.05), but remained significantly poorer than the control site (p < 0.05). Sensation in the central flap areas were similar to peripheral flap areas throughout the follow-up period (p > 0.05). The surrounding skin reached values similar to the control site at 12 months (p > 0.05). Moreover, 50% of patients reported neuropathic pain at 3 months postoperatively, 40% at 6 months, and 0% at 12 months (p < 0.05). CONCLUSION: Mechanical detection, vibration detection, temperature detection, and two-point discrimination significantly improved over time but without reaching normal sensory function at 12 months postoperatively. Neuropathic pain resolved at 12 months.

Role of Oxygen and Its Radicals in Peripheral Nerve Regeneration: From Hypoxia to Physoxia to Hyperoxia.

Oxygen is compulsory for mitochondrial function and energy supply, but it has numerous more nuanced roles. The different roles of oxygen in peripheral nerve regeneration range from energy supply, inflammation, phagocytosis, and oxidative cell destruction in the context of reperfusion injury to crucial redox signaling cascades that are necessary for effective axonal outgrowth. A fine balance between reactive oxygen species production and antioxidant activity draws the line between physiological and pathological nerve regeneration. There is compelling evidence that redox signaling mediated by the Nox family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases plays an important role in peripheral nerve regeneration. Further research is needed to better characterize the role of Nox in physiological and pathological circumstances, but the available data suggest that the modulation of Nox activity fosters great therapeutic potential. One of the promising approaches to enhance nerve regeneration by modulating the redox environment is hyperbaric oxygen therapy. In this review, we highlight the influence of various oxygenation states, i.e., hypoxia, physoxia, and hyperoxia, on peripheral nerve repair and regeneration. We summarize the currently available data and knowledge on the effectiveness of using hyperbaric oxygen therapy to treat nerve injuries and discuss future directions.

Incidence Trends of Melanoma of the Lower Limbs and Hips in the United States: A Surveillance, Epidemiology, and End Results Analysis 2000-2019.

BACKGROUND/AIM: No specific studies on the changes in the incidence of melanoma in the lower limbs and hips have been performed. This article aimed to examine trends in incidence rates of melanoma of the lower extremities in the U.S. PATIENTS AND METHODS: Data from the SEER program provided by the National Cancer Institute were used to examine trends in melanoma incidence from 2000 to 2019. Data analysis was performed from October to December 2022. RESULTS: A total of 192,327 cases of melanoma of the lower limbs and hips were diagnosed from 2000 to 2019 and included in our study. The incidence rate increased from 9.78 to 13.65 cases per 100,000 person-year and by an average annual percent change (AAPC) of 2% (95%CI=1.4-2.9%). The incidence increased by an AAPC of 2.1% in men and 1.7% in women. The incidence among people under 50 remained stable but increased among those over 50 years. Localized stage disease was the only stage where a continuously increasing incidence was observed, with an AAPC of 1.7% (95%CI=0.9-2.5%). Lentiginous melanoma showed the highest incidence trend rate with an AAPC of 2.3% (95%CI=1.0-3.5%). CONCLUSION: The incidence rate of melanoma in the lower limbs and hips increased between 2000 and 2019, with a higher incidence in men, reversing the previously described trend of higher incidence among women. However, incidence among people under 50 remained stable, suggesting the efficacy of prevention campaigns in this population.

A comprehensive review of silk-fibroin hydrogels for cell and drug delivery applications in tissue engineering and regenerative medicine.

Hydrogel scaffolds hold great promise for developing novel treatment strategies in the field of regenerative medicine. Within this context, silk fibroin (SF) has proven to be a versatile material for a wide range of tissue engineering applications owing to its structural and functional properties. In the present review, we report on the design and fabrication of different forms of SF-based scaffolds for tissue regeneration applications, particularly for skin, bone, and neural tissues. In particular, SF hydrogels have emerged as delivery systems for a wide range of bio-actives. Given the growing interest in the field, this review has a primary focus on the fabrication, characterization, and properties of SF hydrogels. We also discuss their potential for the delivery of drugs, stem cells, genes, peptides, and growth factors, including future directions in the field of SF hydrogel scaffolds.

Emerging Strategies for the Biofabrication of Multilayer Composite Amniotic Membranes for Biomedical Applications.

The amniotic membrane (AM) is the innermost part of the fetal placenta, which surrounds and protects the fetus. Due to its structural components (stem cells, growth factors, and proteins), AMs display unique biological properties and are a widely available and cost-effective tissue. As a result, AMs have been used for a century as a natural biocompatible dressing for healing corneal and skin wounds. To further increase its properties and expand its applications, advanced hybrid materials based on AMs have recently been developed. One existing approach is to combine the AM with a secondary material to create composite membranes. This review highlights the increasing development of new multilayer composite-based AMs in recent years and focuses on the benefits of additive manufacturing technologies and electrospinning, the most commonly used strategy, in expanding their use for tissue engineering and clinical applications. The use of AMs and multilayer composite-based AMs in the context of nerve regeneration is particularly emphasized and other tissue engineering applications are also discussed. This review highlights that these electrospun multilayered composite membranes were mainly created using decellularized or de-epithelialized AMs, with both synthetic and natural polymers used as secondary materials. Finally, some suggestions are provided to further enhance the biological and mechanical properties of these composite membranes.

Melanoma of the Lower Limbs and Hips: A Surveillance, Epidemiology, and End Results Analysis of Epidemiology and Survival 2000-2019.

BACKGROUND/AIM: Melanoma, an aggressive skin cancer, poses a significant threat to patients' lives, with lower limbs and hips being among the most affected regions. Epidemiology and survival outcomes of patients with melanoma in the lower extremities were investigated and compared to other sites to better understand tumoral behavior and identify predictors of decreased survival. PATIENTS AND METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was used to search for all skin melanoma cases between 2000 and 2019. Demographic, pathological, and therapeutic factors were compared between affected regions. Overall and disease specific survival were calculated and compared among subgroups. A multivariable analysis was conducted to identify independent prognostic factors. RESULTS: A total of 50,109 patients were diagnosed with melanoma in lower limbs and hips, while 224,121 patients had melanomas in other areas. More women (70.8%) and younger people (mean 55.2 years, SD 16.5) were affected with lower extremities melanoma, with better survival rates than other skin regions. Factors associated with better survival included female sex, younger age, horizontal growth pattern melanomas, and surgery with <1 cm margins. CONCLUSION: Melanoma affecting lower extremities is commonly diagnosed in young females. Prognosis depends on age, stage at diagnosis, and histologic subtype, but remains better compared to other locations.

Mesenchymal Stem Cells in Nerve Tissue Engineering: Bridging Nerve Gap Injuries in Large Animals.

Cell-therapy-based nerve repair strategies hold great promise. In the field, there is an extensive amount of evidence for better regenerative outcomes when using tissue-engineered nerve grafts for bridging severe gap injuries. Although a massive number of studies have been performed using rodents, only a limited number involving nerve injury models of large animals were reported. Nerve injury models mirroring the human nerve size and injury complexity are crucial to direct the further clinical development of advanced therapeutic interventions. Thus, there is a great need for the advancement of research using large animals, which will closely reflect human nerve repair outcomes. Within this context, this review highlights various stem cell-based nerve repair strategies involving large animal models such as pigs, rabbits, dogs, and monkeys, with an emphasis on the limitations and strengths of therapeutic strategy and outcome measurements. Finally, future directions in the field of nerve repair are discussed. Thus, the present review provides valuable knowledge, as well as the current state of information and insights into nerve repair strategies using cell therapies in large animals.

Natural polysaccharides and their derivatives as potential medical materials and drug delivery systems for the treatment of peripheral nerve injuries.

Peripheral nerve repair following injury is one of the most serious problems in neurosurgery. Clinical outcomes are often unsatisfactory and associated with a huge socioeconomic burden. Several studies have revealed the great potential of biodegradable polysaccharides for improving nerve regeneration. We review here the promising therapeutic strategies involving different types of polysaccharides and their bio-active composites for promoting nerve regeneration. Within this context, polysaccharide materials widely used for nerve repair in different forms are highlighted, including nerve guidance conduits, hydrogels, nanofibers and films. While nerve guidance conduits and hydrogels were used as main structural scaffolds, the other forms including nanofibers and films were generally used as additional supporting materials. We also discuss the issues of ease of therapeutic implementation, drug release properties and therapeutic outcomes, together with potential future directions of research.

The Role of Hippo Signaling Pathway and ILK in the Pathophysiology of Human Hypertrophic Scars and Keloids: An Immunohistochemical Investigation.

BACKGROUND: Keloids and hypertrophic scars are characterized by abnormal fibroblast activation and proliferation. While their molecular pathogenesis remains unclear, myofibroblasts have been associated with their development. Hippo pathway effectors YAP/TAZ promote cell proliferation and matrix stiffening. Integrin-linked kinase (ILK), a central component of focal adhesions that mediates cell-matrix interactions, has been linked to tissue repair and fibrosis. The aim of this study was to investigate the expression of key Hippo pathway molecules and ILK in hypertrophic scars and keloids. METHODS: YAP/TAZ, TEAD4, ILK and a-SMA expression were evaluated by immunohistochemistry in keloids (n = 55), hypertrophic scars (n = 38) and normal skin (n = 14). RESULTS: The expression of YAP/TAZ, TEAD4, ILK and a-SMA was higher in fibroblasts of keloids compared to hypertrophic scars while negative in normal skin. There was a significant positive correlation between the expression of ILK and Hippo pathway effectors. CONCLUSIONS: Our results suggest that the deregulation of Hippo signaling and ILK are implicated in keloid and hypertrophic scar formation.

Diagnostic and Therapeutic Roles of the "Omics" in Hypoxic-Ischemic Encephalopathy in Neonates.

Perinatal asphyxia and neonatal encephalopathy remain major causes of neonatal mortality, despite the improved availability of diagnostic and therapeutic tools, contributing to neurological and intellectual disabilities worldwide. An approach using a combination of clinical data, neuroimaging, and biochemical parameters is the current strategy towards the improved diagnosis and prognosis of the outcome in neonatal hypoxic-ischemic encephalopathy (HIE) using bioengineering methods. Traditional biomarkers are of little use in this multifactorial and variable phenotype-presenting clinical condition. Novel systems of biology-based "omics" approaches (genomics, transcriptome proteomics, and metabolomics) may help to identify biomarkers associated with brain and other tissue injuries, predicting the disease severity in HIE. Biomarker studies using omics technologies will likely be a key feature of future neuroprotective treatment methods and will help to assess the successful treatment and long-term efficacy of the intervention. This article reviews the roles of different omics as biomarkers of HIE and outlines the existing knowledge of our current understanding of the clinical use of different omics molecules as novel neonatal brain injury biomarkers, which may lead to improved interventions related to the diagnostic and therapeutic aspects of HIE.

Optimized Decellularization Protocol for Large Peripheral Nerve Segments: Towards Personalized Nerve Bioengineering.

Nerve injuries remain clinically challenging, and allografts showed great promise. Decellularized nerve allografts possess excellent biocompatibility and biological activity. However, the vast majority of decellularization protocols were established for small-size rodent nerves and are not suitable for clinical application. We aimed at developing a new method of decellularizing large-diameter nerves suitable for human transplantation. Repeated rounds of optimization to remove immunogenic material and preserve the extracellular structure were applied to the porcine sciatic nerve. Following optimization, extensive in vitro analysis of the acellular grafts via immunocytochemistry, immunohistology, proteomics and cell transplantation studies were performed. Large segments (up to 8 cm) of the porcine sciatic nerve were efficiently decellularized and histology, microscopy and proteomics analysis showed sufficient preservation of the extracellular matrix, with simultaneous consistent removal of immunogenic material such as myelin, DNA and axons, and axonal growth inhibitory molecules. Cell studies also demonstrated the suitability of these acellular grafts for 3D cell culture studies and translation to future large animal studies and clinical trials. By using non-human donors for peripheral nerve transplantation, significant drawbacks associated with the gold standard can be eliminated while simultaneously preserving the beneficial features of the extracellular matrix.

Comparative hard x-ray tomography for virtual histology of zebrafish larva, human tooth cementum, and porcine nerve.

Purpose: Synchrotron radiation-based tomography yields microanatomical features in human and animal tissues without physical slicing. Recent advances in instrumentation have made laboratory-based phase tomography feasible. We compared the performance of three cutting-edge laboratory systems benchmarked by synchrotron radiation-based tomography for three specimens. As an additional criterion, the user-friendliness of the three microtomography systems was considered. Approach: The three tomography systems-SkyScan 2214 (Bruker-microCT, Kontich, Belgium), Exciscope prototype (Stockholm, Sweden), and Xradia 620 Versa (Zeiss, Oberkochen, Germany)-were given 36 h to measure three medically relevant specimens, namely, zebrafish larva, archaeological human tooth, and porcine nerve. The obtained datasets were registered to the benchmark synchrotron radiation-based tomography from the same specimens and selected ones to the SkyScan 1275 and phoenix nanotom m® laboratory systems to characterize development over the last decade. Results: Next-generation laboratory-based microtomography almost reached the quality achieved by synchrotron-radiation facilities with respect to spatial and density resolution, as indicated by the visualization of the medically relevant microanatomical features. The SkyScan 2214 system and the Exciscope prototype demonstrated the complementarity of phase information by imaging the eyes of the zebrafish larva. The 3 - µ m thin annual layers in the tooth cementum were identified using Xradia 620 Versa. Conclusions: SkyScan 2214 was the simplest system and was well-suited to visualizing the wealth of anatomical features in the zebrafish larva. Data from the Exciscope prototype with the high photon flux from the liquid metal source showed the spiral nature of the myelin sheaths in the porcine nerve. Xradia 620 Versa, with detector optics as typically installed for synchrotron tomography beamlines, enabled the three-dimensional visualization of the zebrafish larva with comparable quality to the synchrotron data and the annual layers in the tooth cementum.

Systematic investigation and comparison of US FDA-approved immunosuppressive drugs FK506, cyclosporine and rapamycin for neuromuscular regeneration following chronic nerve compression injury.

Aim: To compare therapeutic benefits of different immunophilin ligands for treating nerve injuries. Materials & methods: Cyclosporine, FK506 and rapamycin, were evaluated first in vitro on a serum-free culture of embryonic dorsal root ganglia followed by a new in vivo model of chronic nerve compression. Results: Outcomes of the in vitro study have shown a potent effect of cyclosporine and FK506, on dorsal root ganglia axonal outgrowth, comparable to the effect of nerve growth factor. Rapamycin exhibited only a moderate effect. The in vivo study revealed the beneficial effects of cyclosporine, FK506 and rapamycin for neuromuscular regeneration. Cyclosporine showed the better maintenance of the tissues and function. Conclusion: Cyclosporine, FK506 and rapamycin drugs showed potential for treating peripheral nerve chronic compression injuries.

A new model of chronic peripheral nerve compression for basic research and pharmaceutical drug testing.

Aim: To develop a consistent model to standardize research in the field of chronic peripheral nerve neuropathy. Methods: The left sciatic nerve of 8-week-old Sprague-Dawley rats was compressed using a customized instrument leaving a defined post injury nerve lumen (400 µm, 250 µm, 100 µm, 0 µm) for 6 weeks. Sensory and motor outcomes were measured weekly, and histomorphology and electrophysiology after 6 weeks. Results: The findings demonstrated compression depth-dependent sensory and motor pathologies. Quantitative measurements revealed a significant myelin degeneration, axon irregularities and muscle atrophy. At the functional level, we highlighted the dynamics of the different injury profiles. Conclusion: Our novel model of chronic peripheral nerve compression is a useful tool for research on pathophysiology and new therapeutic approaches.

Clinical Studies and Pre-clinical Animal Models on Facial Nerve Preservation, Reconstruction, and Regeneration Following Cerebellopontine Angle Tumor Surgery-A Systematic Review and Future Perspectives.

Background and purpose: Tumorous lesions developing in the cerebellopontine angle (CPA) get into close contact with the 1st (cisternal) and 2nd (meatal) intra-arachnoidal portion of the facial nerve (FN). When surgical damage occurs, commonly known reconstruction strategies are often associated with poor functional recovery. This article aims to provide a systematic overview for translational research by establishing the current evidence on available clinical studies and experimental models reporting on intracranial FN injury. Methods: A systematic literature search of several databases (PubMed, EMBASE, Medline) was performed prior to July 2020. Suitable articles were selected based on predefined eligibility criteria following the Preferred Reporting Items for Systematic Reviews and Meta Analyses (PRISMA) guidelines. Included clinical studies were reviewed and categorized according to the pathology and surgical resection strategy, and experimental studies according to the animal. For anatomical study purposes, perfusion-fixed adult New Zealand white rabbits were used for radiological high-resolution imaging and anatomical dissection of the CPA and periotic skull base. Results: One hundred forty four out of 166 included publications were clinical studies reporting on FN outcomes after CPA-tumor surgery in 19,136 patients. During CPA-tumor surgery, the specific vulnerability of the intracranial FN to stretching and compression more likely leads to neurapraxia or axonotmesis than neurotmesis. Severe FN palsy was reported in 7 to 15 % after vestibular schwannoma surgery, and 6% following the resection of CPA-meningioma. Twenty-two papers reported on experimental studies, out of which only 6 specifically used intracranial FN injury in a rodent (n = 4) or non-rodent model (n = 2). Rats and rabbits offer a feasible model for manipulation of the FN in the CPA, the latter was further confirmed in our study covering the radiological and anatomical analysis of perfusion fixed periotic bones. Conclusion: The particular anatomical and physiological features of the intracranial FN warrant a distinguishment of experimental models for intracranial FN injuries. New Zealand White rabbits might be a very cost-effective and valuable option to test new experimental approaches for intracranial FN regeneration. Flexible and bioactive biomaterials, commonly used in skull base surgery, endowed with trophic and topographical functions, should address the specific needs of intracranial FN injuries.

Schwann Cell-Like Cells: Origin and Usability for Repair and Regeneration of the Peripheral and Central Nervous System.

Functional recovery after neurotmesis, a complete transection of the nerve fiber, is often poor and requires a surgical procedure. Especially for longer gaps (>3 mm), end-to-end suturing of the proximal to the distal part is not possible, thus requiring nerve graft implantation. Artificial nerve grafts, i.e., hollow fibers, hydrogels, chitosan, collagen conduits, and decellularized scaffolds hold promise provided that these structures are populated with Schwann cells (SC) that are widely accepted to promote peripheral and spinal cord regeneration. However, these cells must be collected from the healthy peripheral nerves, resulting in significant time delay for treatment and undesired morbidities for the donors. Therefore, there is a clear need to explore the viable source of cells with a regenerative potential similar to SC. For this, we analyzed the literature for the generation of Schwann cell-like cells (SCLC) from stem cells of different origins (i.e., mesenchymal stem cells, pluripotent stem cells, and genetically programmed somatic cells) and compared their biological performance to promote axonal regeneration. Thus, the present review accounts for current developments in the field of SCLC differentiation, their applications in peripheral and central nervous system injury, and provides insights for future strategies.