Macro and Microfluidic Flows for Skeletal Regenerative Medicine

Fluid flow has a great potential as a cell stimulatory tool for skeletal regenerative medicine, because fluid flow-induced bone cell mechanotransduction in vivo plays a critical role in maintaining healthy bone homeostasis. Applications of fluid flow for skeletal regenerative medicine are reviewed at macro and microscale. Macroflow in two dimensions (2D), in which flow velocity varies along the normal direction to the flow, has explored molecular mechanisms of bone forming cell mechanotransduction responsible for flow-regulated differentiation, mineralized matrix deposition, and stem cell osteogenesis. Though 2D flow set-ups are useful for mechanistic studies due to easiness in in situ and post-flow assays, engineering skeletal tissue constructs should involve three dimensional (3D) flows, e.g., flow through porous scaffolds. Skeletal tissue engineering using 3D flows has produced promising outcomes, but 3D flow conditions (e.g., shear stress vs. chemotransport) and scaffold characteristics should further be tailored. Ideally, data gained from 2D flows may be utilized to engineer improved 3D bone tissue constructs. Recent microfluidics approaches suggest a strong potential to mimic in vivo microscale interstitial flows in bone. Though there have been few microfluidics studies on bone cells, it was demonstrated that microfluidic platform can be used to conduct high throughput screening of bone cell mechanotransduction behavior under biomimicking flow conditions

Nanotopographic Cell Culture Substrate: Polymer-Demixed Nanotextured Films Under Cell Culture Conditions

Modulating physical cell culture environments via nanoscale substrate topographic modification has recently been of significant interest in regenerative medicine. Many studies have utilized a polymer-demixing technique to produce nanotextured films and showed that cellular adhesion, proliferation, and differentiation could be regulated by the shape and scale of the polymer-demixed nanotopographies. However, little attention has been paid to the topographic fidelity of the polymer-demixed films when exposed to cell culture conditions. In this brief article, two polymer-demixing systems were employed to assess topographic changes in polymer-demixed films after fibronectin (FN) extracellular matrix protein adsorption and after incubation in phosphate-buffered saline at 37◦C. We showed that FN adsorption induced very small variations ( \u3c 2 nm) to the polystyrene/polybromostyrene (PS/PBrS)-demixed nanoisland textures, not substantially altering the nanotopographies given by the polymer demixing. In addition, poly(L-lactic acid)/PS (PLLA/PS)-demixed nanoisland topographies using PLLA with Mw = 50 x 103 did not show notable degradation up to day 24

Inducing Neurite Outgrowth by Mechanical Cell Stretch

Establishing extracellular milieus to stimulate neuronal regeneration is a critical need in neuronal tissue engineering. Many studies have used a soluble factor (such as nerve growth factor or retinoic acid [RA]), micropatterned substrate, and electrical stimulation to induce enhanced neurogenesis in neuronal precursor cells. However, little attention has been paid to mechanical stimulation because neuronal cells are not generally recognized as being mechanically functional, a characteristic of mechanoresponsive cells such as osteoblasts, chondrocytes, and muscle cells. In this study, we performed proof-of-concept experiments to demonstrate the potential anabolic effects of mechanical stretch to enhance cellular neurogenesis. We cultured human neuroblastoma (SH-SY5Y) cells on collagen- coated membrane and applied 10% equibiaxial dynamic stretch (0.25 Hz, 120 min/d for 7 days) using a Flexcell device. Interestingly, cell stretch alone, even without a soluble neurogenic stimulatory factor (RA), produced significantly more and longer neurites than the non–RA-treated, static control. Specific neuronal differentiation and cytoskeletal markers (e.g., microtubule-associated protein 2 and neurofilament light chain) displayed compatible variations with respect to stretch stimulation

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration

Prevascularization of 3D printed bone scaffolds by bioactive hydrogels and cell co-culture

Vascularization is a fundamental prerequisite for large bone construct development and remains one of the main challenges of bone tissue engineering. Our current study presents the combination of 3D printing technique with a hydrogel-based prevascularization strategy to generate prevascularized bone constructs. Human adipose derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) were encapsulated within our bioactive hydrogels, and the effects of culture conditions on in vitro vascularization were determined. We further generated composite constructs by forming 3D printed polycaprolactone/hydroxyapatite scaffolds coated with cell-laden hydrogels and determined how the co-culture affected vascularization and osteogenesis. It was demonstrated that 3D co-cultured ADMSC-HUVEC generated capillary-like networks within the porous 3D printed scaffold. The co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis. The prevascularized constructs were subcutaneously implanted into nude mice to evaluate the in vivo vascularization capacity and the functionality of engineered vessels. The hydrogel systems facilitated microvessel and lumen formation and promoted anastomosis of vascular networks of human origin with host murine vasculature. These findings demonstrate the potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects

YAP mechanotransduction under cyclic mechanical stretch loading for mesenchymal stem cell osteogenesis is regulated by ROCK

While yes-associated protein (YAP) is now recognized as a potent mechanosensitive transcriptional regulator to affect cell growth and differentiation including the osteogenic transcription of mesenchymal stem cells (MSCs), most studies have reported the YAP mechanosensing of static mechanophysical cues such as substrate stiffness. We tested MSC response to dynamic loading, i.e., cyclic mechanical stretching, and assessed YAP mechanosensing and resultant MSC osteogenesis. We showed that cyclic stretching at 10% strain and 1 Hz frequency triggered YAP nuclear import in MSCs. YAP phosphorylation at S127 and S397, which is required for YAP cytoplasmic retention, was suppressed by cyclic stretch. We also observed that anti-YAP-regulatory Hippo pathway, LATS phosphorylation, was significantly decreased by stretch. We confirmed the stretch induction of MSC osteogenic transcription and differentiation, and this was impaired under YAP siRNA suggesting a key role of YAP dynamic mechanosensing in MSC osteogenesis. As an underlying mechanism, we showed that the YAP nuclear transport by cyclic stretch was abrogated by ROCK inhibitor, Y27632. ROCK inhibitor also impaired the stretch induction of F-actin formation and MSC osteogenesis, thus implicating the role of the ROCK-F-actin cascade in stretch-YAP dynamic mechanosensing-MSC osteogenesis. Our results provide insight into bone tissue engineering and skeletal regenerative capacity of MSCs especially as regards the role of dynamic mechanical loading control of YAPmediated MSC osteogenic transcription

The LINC complex, mechanotransduction, and mesenchymal stem cell function and fate

Mesenchymal stem cells (MSCs) show tremendous promise as a cell source for tissue engineering and regenerative medicine, and are understood to be mechanosensitive to external mechanical environments. In recent years, increasing evidence points to nuclear envelope proteins as a key player in sensing and relaying mechanical signals in MSCs to modulate cellular form, function, and differentiation. Of particular interest is the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that includes nesprin and SUN. In this review, the way in which cells can sense external mechanical environments through an intact nuclear envelope and LINC complex proteins will be briefly described. Then, we will highlight the current body of literature on the role of the LINC complex in regulating MSC function and fate decision, without and with external mechanical loading conditions. Our review and suggested future perspective may provide a new insight into the understanding of MSC mechanobiology and related functional tissue engineering applications

A Case of Acrodermatitis Enteropathica Localized on the Hands and Feet with a Normal Serum Zinc Level

Acrodermatitis enteropathica is classified as a congenital autosomal recessive type and an acquired transient type. This disease manifests as acral and periorificial dermatitis, alopecia, intractable diarrhea, and failure to thrive. Whereas the autosomal hereditary type is caused by malabsorption of zinc in the intestine, the acquired type is caused by low nutritional support or decreased peripheral release of zinc from blood. We experienced a case of a 5-month old, breast feeding, full-term female presenting with only acral bullous dermatitis without diarrhea, periorificial dermatitis and an abnormal serum zinc level

Identification and characterization of longevity assurance gene related to stress resistance in Brassica

Brassica is a very important vegetable group worldwide and different stresses are a major concern for these crops. Enhancement of resistance against biotic and abiotic stresses by exploiting stress resistance related genes offers the most efficient approach to address this concern. In this study, a stress resistance related gene was identified from the full-length cDNA library of Brassica rapa cv. Osome, which was determined to be Brassica longevity assurance protein (BrLAP) after sequence analysis. A comparison study of this gene showed a high degree of homology with other stress resistance related longevity assurance genes and was shown to be expressed in all organs during all of the developmental growth stages. In addition, this gene significantly responded after cold, drought and ABA stress treatments in Chinese cabbage. All these data revealed that this gene may be involved in plant resistance against stresses.Keywords: Brassica rapa, longevity assurance gene, gene expression, biotic and abiotic stres