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.

Engineering mesenchymal stromal/stem cell-derived extracellular vesicles with improved targeting and therapeutic efficiency for the treatment of central nervous system disorders

Treatment for central nervous system(CNS)disorders is known to be limited by the low regenerative potential of neurons,and thus neurodegenerative insults became known as nearly irreversible ailments.Functional recovery for acquired CNS disorders,such as spinal cord injury(SCI),traumatic brain injury,ischemic stroke,Alzheimer’s disease,Parkinson’s disease,multiple sclerosis(MS),and for congenital CNS abnormalities,such as spina bifida,is not spontaneous and effective treatments are limited to non-existent.Research in the past decades has proven the regenerative potential of stem cells,especially that of mesenchymal stromal/stem cells(MSCs)from various origins,such as bone marrow,placenta,and adipose tissue.

Applications of a new In vivo tumor spheroid based shell-less chorioallantoic membrane 3-D model in bioengineering research

The chicken chorioallantoic membrane (CAM) is a classical in vivo biological model in studies of angiogenesis. Combined with the right tumor system and experimental configuration this classical model can offer new approaches to investigating tumor processes. The increase in development of biotechnolo- gical devices for cancer diagnosis and treatment, calls for more sophisticated tumor models that can easily adapt to the technology, and provide a more accurate, stable and consistent platform for rapid quantitative and qualitative analysis. As we discuss a variety of applications of this novel in vivo tumor spheroid based shell-less CAM model in biomedical engineering research, we will show that it is extremely versatile and easily adaptable to an array of biomedical applications. The model is particularly useful in quantitative studies of the progression of avascular tumors into vascularized tumors in the CAM. Its environment is more stable, flat and has a large working area and wider field of view excellent for imaging and longitudinal studies. Finally, rapid data acquisition, screening and validation of biomedical devices and therapeutics are possible with the short experimental window.

Recent advances and future directions in the management of knee osteoarthritis:Can biological joint reconstruction replace joint arthroplasty and when?

In this article, a concise description of the recent advances in the field of osteoarthritis management is presented. The main focus is to highlight the most promising techniques that emerge in both biological joint replacement and artificial joint arthroplasty. A critical view of high quality evidence regarding outcome and safety profile of these techniques is presented. The potential role of kinematically aligned total knee replacement, navigation, and robotic-assisted surgery is outlined. A critical description of both primary and stem cell-based therapies, the cell homing theory, the use of biologic factors and recent advancements in tissue engineering and regenerative medicine is provided. Based on the current evidence, some thoughts on a realistic approach towards answering these questions are attempted.

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.

Multimodal intravascular imaging technology for characterization of atherosclerosis

Early detection of vulnerable plaques is the critical step in the prevention of acute coronary events.Morphology,composition,and mechanical property of a coronary artery have been demonstrated to be the key characteristics for the identification of vulnerable plaques.Several intravascular multimodal imaging technologies providing co-registered simultaneous images have been developed and applied in clinical studies to improve the characterization of atherosclerosis.In this paper,the authors review the present system and probe designs of representative intra-vascular multimodal techniques.In addition,the scientific innovations,potential limitations,and future directions of these technologies are also discussed.

NON-INVASIVE MEASUREMENT OF DEEP TISSUE TEMPERATURE CHANGES CAUSED BY APOPTOSIS DURING BREAST CANCER NEOADJUVANT CHEMOTHERAPY:A CASE STUDY

Treatment-induced apoptosis of cancer cells is one goal of cancer therapy.Interestingly,more heat is generated by mitochondria during apoptosis,especially the uncoupled apoptotic state,^(1,2) compared to the resting state.In this case study,we explore these thermal effects by longitudinally measuring temperature variations in a breast lesion of a pathological complete responder during neoadjuvant chemotherapy(NAC).Diffuse Optical Spectroscopic Imaging(DOSI)was employed to derive absolute deep tissue temperature using subtle spectral features of the water peak at 975 nm.^(3)A significant temperature increase was observed in time windows during the anthracycline and cyclophosphamide(AC)regimen but not in the paclitaxel and bevacizumab regimen.Hemoglobin concentration changes generally did not follow temperature,suggesting the measured temperature increases were likely due to mitochondrial uncoupling rather than a direct vascular effect.A simultaneous increase of tissue oxygen saturation with temperature was observed,suggesting that oxidative stress also contributes to apoptosis.Although preliminary,this study indicates longitudinal DOSI tissue temperature monitoring provides information that can improve our understanding of the mechanisms of tissue response during NAC.

In utero delivery of mRNA to the heart,diaphragm and muscle with lipid nanoparticles

Nanoparticle-based drug delivery systems have the potential to revolutionize medicine,but their low vascular permeability and rapid clearance by phagocytic cells have limited their medical impact.Nanoparticles delivered at the in utero stage can overcome these key limitations due to the high rate of angiogenesis and cell division in fetal tissue and the under-developed immune system.However,very little is known about nanoparticle drug delivery at the fetal stage of development.In this report,using Ai9 CRE reporter mice,we demonstrate that lipid nanoparticle(LNP)mRNA complexes can deliver mRNA in utero,and can access and transfect major organs,such as the heart,the liver,kidneys,lungs and the gastrointestinal tract with remarkable efficiency and low toxicity.In addition,at 4 weeks after birth,we demonstrate that 50.99±5.05%,36.62±3.42%and 23.7±3.21%of myofiber in the diaphragm,heart and skeletal muscle,respectively,were transfected.Finally,we show here that Cas9 mRNA and sgRNA complexed to LNPs were able to edit the fetal organs in utero.These experiments demonstrate the possibility of non-viral delivery of mRNA to organs outside of the liver in utero,which provides a promising strategy for treating a wide variety of devastating diseases before birth.

Recent advances in lung-on-a-chip technology for modeling respiratory disease

Tissue engineering approaches,including those to functional lung tissues,are finely honed by the inclusion of upgraded devices that mimic biophysical and biochemical features in vivo.Perfusion culture is one of these essential biophysical characteristics enabled by the introduction of microfluidic devices in recent years.This review links the importance of dynamic culture for in vitro maintenance of functional lung cells to the modeling of respiratory disease.We identify and discuss different parameters for fabricating the requisite microfluidic models for lung cells,as well as their application in modeling lung diseases caused by external factors such as smoking and pollution.The possibility of creating a multi-organ-on-a-chip to establish a more physiologically relevant model is highlighted.Overall,the focus is on different prospects for the in vitro modeling approach and for lungs-on-a-chip for developing advanced,reliable technology to analyze the pathophysiology of respiratory diseases and screen potential treatments.

Mesenchymal Stem Cell Allograft Improved Pain Management in Dogs with Osteoarthritis

Background: Osteoarthritis is one of the most common bone diseases, triggered by bone destruction stemming from the inflammatory response of chondrocytes. The disease progresses slowly, but halting its progression or finding a cure remains elusive. The treatment of pain associated with osteoarthritis has yielded unsatisfactory results. In recent years, mesenchymal stem cells (MSCs) have emerged as a potential avenue for addressing the condition. In this study, we used MSCs to treat companion dogs with osteoarthritis. Methods: For this study, 26 animals were included in this study to assess the pain and mobility one month after treatment. The pain scores were obtained from owners using a questionnaire based on the Helsinki Chronic Pain Index, and the Liverpool Osteoarthritis in Dogs (LOAD) Owner questionnaire to assess the mobility of the dogs from stem cell infusion. Results: Questionnaires were administered to dog owners before and one month after treatment, and we found that dogs treated with MSCS experienced an 81.2% ± 6.8% reduction in pain and a 77.9% ± 10.1% increase in mobility, whereas most of the dogs in the untreated control group experienced disease progression. Conclusions: The transplantation of stem cells into companion pets is a promising and expanding opportunity for pet owners with aging and arthritic dogs. MSCs may play an important role in the treatment of OA without complications in companion pets.

Total pancreatectomy and islet autotransplantation: A decade nationwide analysis

AIM: To investigate outcomes and predictors of inhospital morbidity and mortality after total pancreatectomy(TP) and islet autotransplantation. METHODS: The nationwide inpatient sample(NIS) database was used to identify patients who underwent TP and islet autotransplantation(IAT) between 2002-2012 in the United States. Variables of interest were inherent variables of NIS database which included demographic data(age, sex, and race), comorbidities(such as diabetes mellitus, hypertension, and deficiency anemia), and admission type(elective vs nonelective). The primary endpoints were mortality and postoperative complications according to the ICD-9 diagnosis codes which were reported as the second to 25 th diagnosis of patients in the database. Risk adjusted analysis was performed to investigate morbidity predictors. Multivariate regression analysis was used to identify predictors of in-hospital morbidity.RESULTS: We evaluated a total of 923 patients who underwent IAT after pancreatectomy during 2002-2012. Among them, there were 754 patients who had TP + IAT. The most common indication ofsurgery was chronic pancreatitis(86%) followed by acute pancreatitis(12%). The number of patients undergoing TP + IAT annually significantly increased during the 11 years of study from 53 cases in 2002 to 155 cases in 2012. Overall mortality and morbidity of patients were 0% and 57.8 %, respectively. Postsurgical hypoinsulinemia was reported in 42.3% of patients, indicating that 57.7% of patients were insulin independent during hospitalization. Predictors of inhospital morbidity were obesity [adjusted odds ratio(AOR): 3.02, P = 0.01], fluid and electrolyte disorders(AOR: 2.71, P < 0.01), alcohol abuse(AOR: 2.63, P < 0.01), and weight loss(AOR: 2.43, P < 0.01). CONCLUSION: TP + IAT is a safe procedure with no mortality, acceptable morbidity, and achieved high rate of early insulin independence. Obesity is the most significant predictor of in-hospital morbidity.

Functional imaging of neuron-astrocyte interactions in a compartmentalized microfluidic device

Traditional approaches in cultivating neural cells in a dish without orienting their interactions have had only limited success in revealing neural network properties.To enhance the experimental capabilities of studying neural circuitry in vitro,we designed an experimental system combining concepts of micropatterned surfaces,microfluidic devices and genetically encoded biosensors.Micropatterning was used to position neurons and astrocytes in defined locations and guide interactions between the two cell types.Microfluidic chambers were placed atop micropatterned surfaces to allow delivery of different pharmacological agents or viral vectors to the desired cell types.In this device,astrocytes and neurons communicated through grooves molded into the floor of the microfluidic device.By combining microfluidics with genetically encoded calcium indicators as functional readouts,we further demonstrated the utility of this device for analyzing neuron–neuron and neuron–astrocyte interactions in vitro under both healthy and pathophysiological conditions.We found that both spontaneous and evoked calcium dynamics in astrocytes can be modulated by interactions with neurons.In the future,we foresee employing the microdevices described here for studying mechanisms of neurological disorders.

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.

Enhance the delivery of light energy ultra-deep into turbid medium by controlling multiple scattering photons to travel in open channels

Multiple light scattering is considered as the major limitation for deep imaging and focusing in turbid media.In this paper,we present an innovative method to overcome this limitation and enhance the delivery of light energy ultradeep into turbid media with significant improvement in focusing.Our method is based on a wide-field reflection matrix optical coherence tomography(RM-OCT).The time-reversal decomposition of the RM is calibrated with the Tikhonov regularization parameter in order to get more accurate reversal results deep inside the scattering sample.We propose a concept named model energy matrix,which provides a direct mapping of light energy distribution inside the scattering sample.To the best of our knowledge,it is the first time that a method to measure and quantify the distribution of beam intensity inside a scattering sample is demonstrated.By employing the inversion of RM to find the matched wavefront and shaping with a phase-only spatial light modulator,we succeeded in both focusing a beam deep(~9.6 times of scattering mean free path,SMFP)inside the sample and increasing the delivery of light energy by an order of magnitude at an ultra-deep(~14.4 SMFP)position.This technique provides a powerful tool to understand the propagation of photon in a scattering medium and opens a new way to focus light inside biological tissues.

Cell-free systems in the new age of synthetic biology

合成生物学的来临迎来了没有房间的抄写翻译系统的新应用程序。这些没有房间的系统用细胞的蛋白质被重新组成，并且对他们的作文的模块化的控制顺从。这里，我们在合成基因电路和部件的快速的设计讨论没有房间的系统，以及他们的新应用的历史的前进， biomolecules，传染疾病的诊断，和疫苗的合成的指导进化。最后，我们在场我们没有房间的合成生物学的未来方向上的视觉。

Fabrication of all-transparent polymer-based and encapsulated nanofluidic devices using nano-indentation lithography

In this paper,we describe a novel and simple process for the fabrication of all-transparent and encapsulated polymeric nanofluidic devices using nano-indentation lithography.First,a nanomechanical probe is used to‘scratch’nanoscale channels on polymethylmethacrylate(PMMA)substrates with sufficiently high hardness.Next,polydimethylsiloxane(PDMS)is used twice to duplicate the nanochannels onto PDMS substrates from the‘nano-scratched’PMMA substrates.A number of experiments are conducted to explore the relationships between the nano-indentation parameters and the nanochannel dimensions and to control the aspect ratio of the fabricated nanochannels.In addition,traditional photolithography combined with soft lithography is employed to fabricate microchannels on another PDMS‘cap’substrate.After manually aligning the substrates,all uncovered channels on two separate PDMS substrates are bonded to achieve a sealed and transparent nanofluidic device,which makes the dimensional transition from microscale to nanoscale feasible.The smallest dimensions of the achievable nanochannels that we have demonstrated thus far are of~20 nm depth and~800 nm width,with lengths extendable beyond 100μm.Fluid flow experiments are performed to verify the reliability of the device.Two types of colloidal solution are used to visualize the fluid flow through the nanochannels,that is,ethanol is mixed with gold colloid or fluorescent dye(fluorescein isothiocyanate),and the flow rate and filling time of liquid in the nanochannels are estimated based on time-lapsed image data.The simplicity of the fabrication process,bio-compatibility of the polymer substrates,and optical transparency of the nanochannels for flow visualization are key characteristics of this approach that will be very useful for nanofluidic and biomolecular research applications in the future.

Whole-blood sorting,enrichment and in situ immunolabeling of cellular subsets using acoustic microstreaming

Analyzing undiluted whole human blood is a challenge due to its complex composition of hematopoietic cellular populations,nucleic acids,metabolites,and proteins.We present a novel multi-functional microfluidic acoustic streaming platform that enables sorting,enrichment and in situ identification of cellular subsets from whole blood.This single device platform,based on lateral cavity acoustic transducers(LCAT),enables(1)the sorting of undiluted donor whole blood into its cellular subsets(platelets,RBCs,and WBCs),(2)the enrichment and retrieval of breast cancer cells(MCF-7)spiked in donor whole blood at rare cell relevant concentrations(10 mL^(−1)),and(3)on-chip immunofluorescent labeling for the detection of specific target cellular populations by their known marker expression patterns.Our approach thus demonstrates a compact system that integrates upstream sample processing with downstream separation/enrichment,to carry out multi-parametric cell analysis for blood-based diagnosis and liquid biopsy blood sampling.

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.

A modular microfluidic system based on a multilayered configuration to generate large-scale perfusable microvascular networks

The vascular network of the circulatory system plays a vital role in maintaining homeostasis in the human body.In this paper,a novel modular microfluidic system with a vertical two-layered configuration is developed to generate largescale perfused microvascular networks in vitro.The two-layer polydimethylsiloxane(PDMS)configuration allows the tissue chambers and medium channels not only to be designed and fabricated independently but also to be aligned and bonded accordingly.This method can produce a modular microfluidic system that has high flexibility and scalability to design an integrated platform with multiple perfused vascularized tissues with high densities.The medium channel was designed with a rhombic shape and fabricated to be semiclosed to form a capillary burst valve in the vertical direction,serving as the interface between the medium channels and tissue chambers.Angiogenesis and anastomosis at the vertical interface were successfully achieved by using different combinations of tissue chambers and medium channels.Various large-scale microvascular networks were generated and quantified in terms of vessel length and density.Minimal leakage of the perfused 70-kDa FITC-dextran confirmed the lumenization of the microvascular networks and the formation of tight vertical interconnections between the microvascular networks and medium channels in different structural layers.This platform enables the culturing of interconnected,large-scale perfused vascularized tissue networks with high density and scalability for a wide range of multiorgan-on-a-chip applications,including basic biological studies and drug screening.

Optical force-induced nonlinearity and self-guiding of light in human red blood cell suspensions

Osmotic conditions play an important role in the cell properties of human red blood cells(RBCs),which are crucial for the pathological analysis of some blood diseases such as malaria.Over the past decades,numerous efforts have mainly focused on the study of the RBC biomechanical properties that arise from the unique deformability of erythrocytes.Here,we demonstrate nonlinear optical effects from human RBCs suspended in different osmotic solutions.Specifically,we observe self-trapping and scattering-resistant nonlinear propagation of a laser beam through RBC suspensions under all three osmotic conditions,where the strength of the optical nonlinearity increases with osmotic pressure on the cells.This tunable nonlinearity is attributed to optical forces,particularly the forward-scattering and gradient forces.Interestingly,in aged blood samples(with lysed cells),a notably different nonlinear behavior is observed due to the presence of free hemoglobin.We use a theoretical model with an optical force-mediated nonlocal nonlinearity to explain the experimental observations.Our work on light self-guiding through scattering biosoft-matter may introduce new photonic tools for noninvasive biomedical imaging and medical diagnosis.