Stem cell-based in utero therapies for spina bifida: implications for neural regeneration

The history: Myelomeningocele - also known as spina bifida- is a devastating congenital anomaly of the central nervoussystem that is caused by the malformation of the spinal cordand vertebral column during embryogenesis. Depending onthe location of the spina bifida lesion on the spine, patientssuffer from neurological dysfunction ranging from paresisand incontinence to complete paralysis. The current standardof care for spina bifida is in utero surgical repair of the defect,which has been shown to minimize the secondary deficits associatedwith this disorder (Adzick et al., 2011). Despite thesesuccesses, this approach does not reliably improve neurologicfunction of affected children. Several groups, including ourown, have performed studies aimed at augmenting the inutero surgical repair of spina bifida by applying principlesof stem cell and tissue engineering to provide an enhancedprotection of the exposed neural elements (Saadai et al., 2011,2013; Wang et al., 2015; Brown et al., 2016). The ultimategoal of these studies is to improve the neurologic function inpatients while maintaining the benefits of the existing fetalsurgical treatment.

Clonal isolation of endothelial colony-forming cells from early gestation chorionic villi of human placenta for fetal tissue regeneration

BACKGROUND Endothelial colony-forming cells(ECFCs)have been implicated in the process of vascularization,which includes vasculogenesis and angiogenesis.Vasculogenesis is a de novo formation of blood vessels,and is an essential physiological process that occurs during embryonic development and tissue regeneration.Angiogenesis is the growth of new capillaries from pre-existing blood vessels,which is observed both prenatally and postnatally.The placenta is an organ composed of a variety of fetal-derived cells,including ECFCs,and therefore has significant potential as a source of fetal ECFCs for tissue engineering.AIM To investigate the possibility of isolating clonal ECFCs from human early gestation chorionic villi(CV-ECFCs)of the placenta,and assess their potential for tissue engineering.METHODS The early gestation chorionic villus tissue was dissociated by enzyme digestion.Cells expressing CD31 were selected using magnetic-activated cell sorting,and plated in endothelial-specific growth medium.After 2-3 wks in culture,colonies displaying cobblestone-like morphology were manually picked using cloning cylinders.We characterized CV-ECFCs by flow cytometry,immunophenotyping,tube formation assay,and Dil-Ac-LDL uptake assay.Viral transduction of CVECFCs was performed using a Luciferase/tdTomato-containing lentiviral vector,and transduction efficiency was tested by fluorescent microscopy and flow cytometry.Compatibility of CV-ECFCs with a delivery vehicle was determined using an FDA approved,small intestinal submucosa extracellular matrix scaffold.RESULTS After four passages in 6-8 wks of culture,we obtained a total number of 1.8×107 CV-ECFCs using 100 mg of early gestational chorionic villus tissue.Immunophenotypic analyses by flow cytometry demonstrated that CV-ECFCs highly expressed the endothelial markers CD31,CD144,CD146,CD105,CD309,only partially expressed CD34,and did not express CD45 and CD90.CV-ECFCs were capable of acetylated low-density lipoprotein uptake and tube formation,similar to cord blood-derived ECFCs(CB-ECFCs).CV-ECFCs can be transduced with a Luciferase/tdTomato-containing lentiviral vector at a transduction efficiency of 85.1%.Seeding CV-ECFCs on a small intestinal submucosa extracellular matrix scaffold confirmed that CV-ECFCs were compatible with the biomaterial scaffold.CONCLUSION In summary,we established a magnetic sorting-assisted clonal isolation approach to derive CV-ECFCs.A substantial number of CV-ECFCs can be obtained within a short time frame,representing a promising novel source of ECFCs for fetal treatments.

Targeting angiogenesis for fracture nonunion treatment in inflammatory disease

Atrophic fracture nonunion poses a significant clinical problem with limited therapeutic interventions.In this study,we developed a unique nonunion model with high clinical relevance using serum transfer-induced rheumatoid arthritis(RA).Arthritic mice displayed fracture nonunion with the absence of fracture callus,diminished angiogenesis and fibrotic scar tissue formation leading to the failure of biomechanical properties,representing the major manifestations of atrophic nonunion in the clinic.Mechanistically,we demonstrated that the angiogenesis defect observed in RA mice was due to the downregulation of SPP1 and CXCL12 in chondrocytes,as evidenced by the restoration of angiogenesis upon SPP1 and CXCL12 treatment in vitro.In this regard,we developed a biodegradable scaffold loaded with SPP1 and CXCL12,which displayed a beneficial effect on angiogenesis and fracture repair in mice despite the presence of inflammation.Hence,these findings strongly suggest that the sustained release of SPP1 and CXCL12 represents an effective therapeutic approach to treat impaired angiogenesis and fracture nonunion under inflammatory conditions.

Astrocyte evolution and human specificity

The cerebral cortex is one of the most complex structures of the mammalian central nervous system and accounts for the extraordinary cognitive abilities in primates and humans.Since the 19th century,neur ons have been believed to be the main players in the building of the brain,yet astrocytes also play a crucial role as fundamental building blocks of the cerebral cortex complexity.

Convergence of human and veterinary medicine:leveraging canine naturally occurring neurological disorders to develop regenerative treatments

In recent years,large animal models of naturally occurring diseases have become increasingly studied,with the rationale that their disease attributes may better recapitulate the pathological features of corresponding human diseases as compared to induced disease models(Hoffman and Dow,2016).Of the available naturally occurring disease models,the canine is increasingly recognized as a valuable pre-clinical animal model in translational medicine for numerous human diseases,including cancer.

RhoA as a target to promote neuronal survival and axon regeneration

Paralysis following spinal cord injury （SCI） is due to failure of axonal regeneration. It is believed that the capacities of neurons to regrow their axons are due partly to their intrinsic characteristics, which in turn are greatly influenced by several types of inhibitory molecules that are present, or even increased in the extracellular environment of the injured spinal cord. Many of these inhibitory molecules have been studied extensively in recent years. It has been suggested that the small GTPase RhoA is an intracellular convergence point for signaling by these extracellular inhibitory molecules, but due to the complexity of the central nervous system （CNS） in mammals, and the limitation of pharmacological tools, the specific roles of RhoA are unclear. By exploiting the anatomical and technical advantages of the lamprey CNS, we recently demonstrated that RhoA knockdown promotes true axon regeneration through the lesion site after SCI. In addition, we found that RhoA knockdown protects the large, identified reticulospinal neurons from apoptosis after their axons were axotomized in spinal cord. Therefore, manipulation of the RhoA signaling pathway may be an important approach in the development of treatments that are both neuroprotective and axon regeneration-promoting, to enhance functional recovery after SCI.

Empowering sonic hedgehog to rescue brain cells after ischemic stroke

Ischemic stroke occurs when blood supply to the brain is interrupted. This can cause irreversible injury to the cen- tral nervous system （CNS） tissue. Each year in the United States almost 800,000 people experience a new or recur- rent stroke. 15% of stroke patients die shortly after insult and only 10% recover completely, leaving the majority of surviving stroke patients with disabilities. Tissue-type plasminogen activator （tPA） is the only available therapy for stroke but its clinical use is limited because of associ- ated danger of intracranial hemorrhage. Therefore, there is an emergent need for stroke therapeutics that are safe and effective when administered at a later time point after insult.

Exosomal microRNAs from mesenchymal stem/stromal cells: Biology and applications in neuroprotection

Mesenchymal stem/stromal cells(MSCs)are extensively studied as cell-therapy agents for neurological diseases.Recent studies consider exosomes secreted by MSCs as important mediators for MSCs’neuroprotective functions.Exosomes transfer functional molecules including proteins,lipids,metabolites,DNAs,and coding and non-coding RNAs from MSCs to their target cells.Emerging evidence shows that exosomal microRNAs(miRNAs)play a key role in the neuroprotective properties of these exosomes by targeting several genes and regulating various biological processes.Multiple exosomal miRNAs have been identified to have neuroprotective effects by promoting neurogenesis,neurite remodeling and survival,and neuroplasticity.Thus,exosomal miRNAs have significant therapeutic potential for neurological disorders such as stroke,traumatic brain injury,and neuroinflammatory or neurodegenerative diseases and disorders.This review discusses the neuroprotective effects of selected miRNAs(miR-21,miR-17-92,miR-133,miR-138,miR-124,miR-30,miR146a,and miR-29b)and explores their mechanisms of action and applications for the treatment of various neurological disease and disorders.It also provides an overview of state-of-the-art bioengineering approaches for isolating exosomes,optimizing their yield and manipulating the miRNA content of their cargo to improve their therapeutic potential.

IκB-ζsignaling promotes chondrocyte inflammatory phenotype,senescence,and erosive joint pathology

Osteoarthritis is a joint disease characterized by a poorly-defined inflammatory response that does not encompass a massive immune cell infiltration yet contributes to cartilage degradation and loss of joint mobility, suggesting a chondrocyte intrinsic inflammatory response. Using primary chondrocytes from joints of osteoarthritic mice and patients, we first show that these cells express ample pro-inflammatory markers and RANKL in an NF-κB dependent manner. The inflammatory phenotype of chondrocytes was recapitulated by exposure of chondrocytes to IL-1β and bone particles, which were used to model bone matrix breakdown products revealed to be present in synovial fluid of OA patients, albeit their role was not defined. We further show that bone particles and IL-1β can promote senescent and apoptotic changes in primary chondrocytes due to oxidative stress from various cellular sources such as the mitochondria. Finally, we provide evidence that inflammation, oxidative stress and senescence converge upon IκB-ζ, the principal mediator downstream of NF-κB, which regulates expression of RANKL, inflammatory, catabolic,and SASP genes. Overall, this work highlights the capacity and mechanisms by which inflammatory cues, primarily joint degradation products, i.e., bone matrix particles in concert with IL-1β in the joint microenvironment, program chondrocytes into an “inflammatory phenotype” which inflects local tissue damage.

Disruption of bone and skeletal muscle in severe burns

Severe burn injury triggers the body＇s nonspecific adaptive responses to acute insult, including the systemic inflammatory and stress responses, as well as the sympathetic response to immobilization. These responses trigger inflammatory bone resorption followed by glucocorticoid-induced apoptosis of osteoblasts and probably osteocytes. Because these patients are catabolic, they suffer concomitant muscle wasting and negative nitrogen balance. The use of anabolic agents such as recombinant human growth hormone and oxandrolone results in improved bone mineral content and muscle strength after approximately I year. Use of bisphosphonates within the first 10 days of a severe burn completely blocks the resorptive bone loss and has the added advantage of appearing to preserve muscle protein from excessive breakdown. The mechanism for the protective effect on muscle is not currently known. However, if the effect of bisphosphonates on muscle can be confirmed, it raises the possibility that bone communicates with muscle.

Mitochondrial translocator protein(TSPO),astrocytes and neuroinflammation

Mitochondrial translocator protein（TSPO）has been a somewhat mysterious player in searching for its true biological functions.TSPO is involved in mitochondrial cholesterol transport and steroids biosynthesis and predominantly expressed in steroid-synthesizing tissues including the central nervous system（CNS）.However,

“手风琴”牵张成骨的实验和模拟研究

目的牵张成骨术(DO)通过牵张器对骨骼施加稳定和缓慢的牵张力,刺激截骨间隙内新骨形成和矿化,以实现延长骨骼的目的。但其最主要的临床问题是巩固期新骨形成缓慢,导致治疗周期漫长。临床报道,在巩固期施加交替牵-模式的"手风琴"技术(AT)可有效治疗一些骨再生不良或矿化缓慢的情况。然而,尚不清楚AT的最佳应用时机。采用动物实验与数值模拟相结合的方法,探究在巩固期不同时机行使AT对DO骨再生的影响。方法对小鼠股骨干进行截骨术和为期10 d的牵张。

Allergic dermatitis after knee arthroscopy with repeated exposure to Dermabond Prineo^(TM) in pediatric patients:Two case reports

BACKGROUND Allergic contact dermatitis(ACD)secondary to Dermabond Prineo^(TM) is rare,but documented.To our knowledge,there are no described reports of this ACD reaction within the pediatric population following arthroscopic surgery.CASE SUMMARY We report two cases of pediatric ACD upon second exposure to Dermabond Prineo^(TM) after knee arthroscopy.Both cases presented within two weeks of the inciting second exposure.The cases resolved with differing described combinations of sterile cleaning,diphenhydramine,and antibiotic administration.No long-term sequelae were found.CONCLUSION This case report elucidates the rare complication of allergic dermatitis secondary to Dermabond Prineo^(TM) repeat exposure use in pediatric arthroscopy.

A bio-instructive parylene-based conformal coating suppresses thrombosis and intimal hyperplasia of implantable vascular devices

Implantable vascular devices are widely used in clinical treatments for various vascular diseases. However, current approved clinical implantable vascular devices generally have high failure rates primarily due to their surface lacking inherent functional endothelium. Here, inspired by the pathological mechanisms of vascular device failure and physiological functions of native endothelium, we developed a new generation of bioactive parylene (poly(p-xylylene))-based conformal coating to address these challenges of the vascular devices. This coating used a polyethylene glycol (PEG) linker to introduce an endothelial progenitor cell (EPC) specific binding ligand LXW7 (cGRGDdvc) onto the vascular devices for preventing platelet adhesion and selectively capturing endogenous EPCs. Also, we confirmed the long-term stability and function of this coating in human serum. Using two vascular disease-related large animal models, a porcine carotid artery interposition model and a porcine carotid artery-jugular vein arteriovenous graft model, we demonstrated that this coating enabled rapid generation of self-renewable “living” endothelium on the blood contacting surface of the expanded polytetrafluoroethylene (ePTFE) grafts after implantation. We expect this easy-to-apply conformal coating will present a promising avenue to engineer surface properties of “off-the-shelf” implantable vascular devices for long-lasting performance in the clinical settings.

PhaseFIT:live-organoid phase-fluorescent image transformation via generative AI

Organoid models have provided a powerful platform for mechanistic investigations into fundamental biological processes involved in the development and function of organs.Despite the potential for image-based phenotypic quantification of organoids,their complex 3D structure,and the time-consuming and labor-intensive nature of immunofluorescent staining present significant challenges.In this work,we developed a virtual painting system,PhaseFIT(phase-fluorescent image transformation)utilizing customized and morphologically rich 2.5D intestinal organoids,which generate virtual fluorescent images for phenotypic quantification via accessible and low-cost organoid phase images.This system is driven by a novel segmentation-informed deep generative model that specializes in segmenting overlap and proximity between objects.The model enables an annotation-free digital transformation from phase-contrast to multi-channel fluorescent images.The virtual painting results of nuclei,secretory cell markers,and stem cells demonstrate that PhaseFIT outperforms the existing deep learning-based stain transformation models by generating fine-grained visual content.We further validated the efficiency and accuracy of PhaseFIT to quantify the impacts of three compounds on crypt formation,cell population,and cell stemness.PhaseFIT is the first deep learning-enabled virtual painting system focused on live organoids,enabling large-scale,informative,and efficient organoid phenotypic quantification.PhaseFIT would enable the use of organoids in high-throughput drug screening applications.

Hydrogels for the management of second-degree burns:currently available options and future promise

Burn wounds result from exposure to hot liquids,chemicals,fire,electric discharge or radiation.Wound severity ranges from first-degree injury,which is superficial,to fourth-degree injury,which exposes bone,tendons and muscles.Rapid assessment of burn depth and accurate wound management in the outpatient setting is critical to prevent injury progression into deeper layers of the dermis.Injury progression is of particular pertinence to second-degree burns,which are the most common form of thermal burn.As our understanding of wound healing advances,treatment options and technologies for second-degree burn management also evolve.Polymeric hydrogels are a class of burn wound dressings that adhere to tissue,absorb wound exudate,protect from the environment,can be transparent facilitating serial wound evaluation and,in some cases,enable facile removal for dressing changes.This review briefly describes the burn level classification and common,commercially available dressings used to treat second-degree burns,and then focuses on new polymeric hydrogel burn dressings under preclinical development analyzing their design,structure and performance.The review presents the follow key learning points:(1)introduction to the integument system and the wound-healing process;(2)classification of burns according to severity and clinical appearance;(3)available dressings currently used for second-degree burns;(4)introduction to hydrogels and their preparation and characterization techniques;and(5)pre-clinical hydrogel burn wound dressings currently being developed.

Layer-by-layer functionalized nanotube arrays:A versatile microfluidic platform for biodetection

We demonstrate the layer-by-layer(LbL)assembly of polyelectrolyte multilayers(PEM)on three-dimensional nanofiber scaffolds.High porosity(99%)aligned carbon nanotube(CNT)arrays are photolithographically patterned into elements that act as textured scaffolds for the creation of functionally coated(nano)porous materials.Nanometer-scale bilayers of poly(allylamine hydrochloride)/poly(styrene sulfonate)(PAH/SPS)are formed conformally on the individual nanotubes by repeated deposition from aqueous solution in microfluidic channels.Computational and experimental results show that the LbL deposition is dominated by the diffusive transport of the polymeric constituents,and we use this understanding to demonstrate spatial tailoring on the patterned nanoporous elements.A proof-of-principle application,microfluidic bioparticle capture using N-hydroxysuccinimide-biotin binding for the isolation of prostate-specific antigen(PSA),is demonstrated.

Tissue engineering of skin and regenerative medicine for wound care

Engineering of biologic skin substitutes has progressed over time from individual applications of skin cells, or biopolymer scaffolds, to combinations of cells and scaffolds for treatment, healing, and closure of acute and chronic skin wounds. Skin substitutes may be categorized into three groups: acellular scaffolds, temporary substitutes containing allogeneic skin cells, and permanent substitutes containing autologous skin cells. Combined use of acellular dermal substitutes with permanent skin substitutes containing autologous cells has been shown to provide definitive wound closure in burns involving greater than 90% of the total body surface area. These advances have contributed to reduced morbidity and mortality from both acute and chronic wounds but, to date, have failed to replace all of the structures and functions of the skin. Among the remaining deficiencies in cellular or biologic skin substitutes are hypopigmentation, absence of stable vascular and lymphatic networks, absence of hair follicles, sebaceous and sweat glands, and incomplete innervation. Correction of these deficiencies depends on regulation of biologic pathways of embryonic and fetal development to restore the full anatomy and physiology of uninjured skin. Elucidation and integration of developmental biology into future models of biologic skin substitutes promises to restore complete anatomy and physiology, and further reduce morbidity from skin wounds and scar. This article offers a review of recent advances in skin cell thrapies and discusses the future prospects in cutaneous regeneration.

Children are not little adults:blood transfusion in children with burn injury

Blood transfusion in burns larger than 20%total body surface area (TBSA) are frequent due to operative procedures, blood sampling, and physiologic response to burn injury. Optimizing the use of blood transfusions requires an understanding of the physiology of burn injury, the risks and benefits of blood transfusion, and the indications for transfusion. Age also plays a role in determining blood transfusion requirements. Children in particular have a different physiology than adults, which needs to be considered prior to transfusing blood and blood products. This article describes the physiologic differences between children and adults in general and after burn injury and describes how these differences impact blood transfusion practices in children.

Considerations for pediatric burn sedation and analgesia

Burn patients experience anxiety and pain in the course of their injury, treatment, and recovery. Hence, treatment of anxiety and pain is paramount after burn injury. Children, in particular, pose challenges in anxiety and pain management due to their unique physiologic, psychologic, and anatomic status. Burn injuries further complicate pain management and sedation as such injuries can have effects on medication response and elimination. Burn injuries further complicate pain management and sedation as such injuries can have effects on medication response and elimination. The purpose of this review is to describe the challenges associated with management of anxiety, pain, and sedation in burned children and to describe the different options for treatment of anxiety and pain in burned children.