The production and the experimental work presented herein reflect the devoted and enthusiastic help of Reynaldo Pareja. Funded by Colombia's Administrative Department of Science, Technology and Innovation (Colciencias) and by a grant from the Bill &amp; Melinda Gates Foundation through the Grand Challenges Explorations initiative.

1. Plants
<ol>
 <li>Surface-sterilize bean seeds (cv. Calima) by immersing them for two minutes in 0.5% sodium hypochlorite and two minutes in 70% ethanol. Rinse the seeds three times in sterile distilled water.</li>
 <li>Evaluate the success of your sterilization by plating 100 &mu;l of the last rinsing water on potato dextroxe agar (PDA) media, and incubating the plate for 10 days at 25 &deg;C. Terminate and restart the experiment if any growth is seen on the plate. </li>
 <li>Plant the seeds in groups of three in pots containing a sterile mixture of soil and sand at a 2:1 ratio. Transfer the pots to a growth chamber at 25 &deg;C, ca. 50% RH and 12 hr photoperiod. One week after germination, eliminate the two least vigorous seedlings. Water every 2-3 days with sterile distilled water and fertilize 10 and 20 days after planting with a 6 g/L water solution of NPK 15-15-15 fertilizer. </li>
</ol>
2. Fungus
<ol>
 <li>Obtain a commercial formulation of Beauveria bassiana strain GHA (Mycotrol SE, Laverlam, Cali, Colombia). </li>
 <li>To generate a single-spore stock culture, suspend ca. one inoculating loop full of conidia in 1 ml of a 0.1% water solution of Triton-X 100 and vortex for 10 sec. Then, plate 100 &mu;l of the suspension on 2.5% Noble agar and incubate for 24 hr at 25 &deg;C. Transfer a single germinating conidium into a 100 mm plate containing PDA and grow until it covers the entire plate (ca. 3-4 weeks).</li>
 <li>Under sterile conditions, scrape the fungal growth from the surface of the medium and suspend it in 10 ml sterile 0.1% Triton-X 100. Vortex for one minute. Then, filter the suspension through a sterile cheesecloth to remove hyphae and obtain the stock suspension. </li>
 <li>Use a hemocytometer to estimate the conidial concentration of the stock suspension. To facilitate conidial counts, prepare a 10,000-fold serial dilution of the stock, each time transferring 100 &mu;l of conidial suspension in 900 &mu;l of 0.1% Triton-X 100, and vortexing for 10 sec before the next dilution. </li>
 <li>To generate the inoculum, adjust the stock suspension to a final concentration of 108 conidia/ml, using the formula: 
 <img src="/files/ftp_upload/50360/50360eq1.jpg" alt="equation 1" fo:src="/files/ftp_upload/50360/50360eq1highres.jpg"/> </li>
 <li>To assess conidial viability, plate 100 &mu;l of the 10,000-fold dilution on 2.5% Noble agar and incubate for 24 hr at 25 &deg;C. Then, inspect three random groups of 100 conidia to estimate percent germination. Consider a conidium germinated when a visible germ tube longer than half the diameter of the conidium projects from it. Only use the suspension when the average percent germination exceeds 90%. </li>
</ol>
3. Inoculation
<ol>
 <li>Inoculate plants when they reach their first true leaf stage (ca. 14 days after planting). Water plants to soil capacity with sterile distilled water 24 hr before inoculations.</li>
 <li>For the foliar spray method, use a manual atomizer to apply the conidial suspension (treatment) or 0.1% Triton-X 100 (control) to the adaxial (upper) surface of leaves until they reach saturation. Cover the top of the pot with aluminum foil to avoid conidial runoff to the soil. After spraying, cover plants with a plastic bag for 24 hr to maintain a high level of humidity facilitating fungal invasion. </li>
 <li>For the soil drench method, use a graduated cylinder to apply 10 ml of conidial suspension (treatment) or 0.1% Triton-X 100 (control) to the surface of the soil at the base of the plant. </li>
 <li>After inoculations, return plants to the growth chambers arranging them in a randomized complete block design. No less than four additional experimental blocks should be installed to allow evaluations of plant growth, in addition to evaluations of endophytic colonization, in the same experiment. </li>
</ol>
4. Evaluations
<ol>
 <li>Evaluate the experiment one block at a time, selecting the blocks in random order. This is particularly important for large experiments that cannot be evaluated on a single day.</li>
 <li>Before processing a plant, measure and record its height from the base to the apical meristem. Then, uproot carefully and wash thoroughly in running tap water. </li>
 <li>From each plant, sample two leaflets, two pieces of root and two pieces of stem. Select leaflet samples randomly from the first true leaf of the plant. Then, obtain two stem samples, 3 cm-long each, from the middle of the plant and from near the soil surface. Finally, obtain two taproot samples, also 3 cm-long each, from the middle of the root and from 1 cm behind the root tip. Place the samples on three separate paper bags and label appropriately. </li>
 <li>After washing and sampling all the plants in a block, start by processing the leaves, then roots, and finally the stems.</li>
 <li>Surface sterilize tissues in a sterile laminar flow hood as in 1.1, above. Rinse each sample three times by immersion in sterile distilled water and let it dry in sterile towel paper. Then, dissect and discard its outer edges, where endophytes might have been eliminated due to contact with disinfectants.</li>
 <li>Cut the trimmed sample into six sections, averaging 6x6 mm for leaves and 6 mm-long for stems and roots. Plate the six sections on a 60 mm Petri plate with PDA media supplemented with the antibiotics tetracycline, streptomycin and penicillin at 2 mg/L each. Seal the plate with parafilm and incubate in the dark at 25 &deg;C. Each plant yields six plates, two per plant part. </li>
 <li>Change the rinse water after processing each block of a given plant part. Before discarding the used rinsing water, plate a 100 &mu;l sample on PDA media and incubate for 10 days at 25 &deg;C to assess sterilization success. If fungal growth ensues, do not consider the corresponding samples for analyses. </li>
 <li>Inspect the plates every 2-3 days for 20 days to observe and record fungal growth. Excise and transfer plant sections exhibiting presence of fungal endophytes to plates containing fresh PDA. This will avoid contamination of neighboring plant sections in the original plate. </li>
 <li>Record B. bassiana growth from plant sections. Beauveria bassiana can be identified by characteristic white dense mycelia becoming cream to pale yellow at the edge. When in doubt, mount the specimen in a drop of water and inspect under a microscope, looking for globose conidia and zigzag-shaped conidiophores, characteristic of the species. </li>
 <li>Use additional experimental blocks to evaluate the impact of the treatments on plant biomass. First, measure their height from the base to the top of the apical meristem. Then, carefully uproot and wash plants in tap water and let them dry at 45 &deg;C for three days to determine their dry weight. </li>
</ol>

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The authors declare that they have no competing financial interests.

Many factors can influence the specific outcome of an experiment to establish a fungal entomopathogen as an endophyte. Our results demonstrate the inoculation method is one of them. Biological factors to experiment with include the crop species or cultivar selected and the fungal entomopathogen species strain or isolate used. Other factors to consider manipulating include the concentration of the inoculum, the age of the plant during inoculations, and the plant's growing conditions. 
It would be ideal for an inoculation method to result in systemic plant colonization by a fungal entomopathogen14,17,18,21. Instead, it appears that inoculation methods tend to favor a specific pattern of local colonization. In coffee, for example, foliar sprays favor leaf colonization whereas soil drenches favor root colonization17. We found the same pattern in the common bean. Ultimately, the choice of inoculation method should be guided by the intended location of the endophyte within a plant, presumably matching the niche of the target herbivore or plant pathogen. 
Although commonly used, endophyte detection and quantification based on media cultures can be costly, difficult and error prone. For example, a total of 10,800 plant sections (plated on 1,800 Petri dishes) were evaluated in an experiment to optimize B. bassiana inoculations on banana16. Out of these, 4,496 sections were colonized by a putative B. bassiana, as identified mainly by colony morphology. Clearly, a microscopic verification of the species for each colony would have been a desirable but unaffordable step. On the other hand, 1,176 sections were colonized by other fungi and were discarded and treated as missing data16. The probability exists, however, that B. bassiana was a poor competitor or slower to grow, and could have eventually emerged from those sections if allowed sufficient time. Therefore, endophyte detection methods based on media cultures are subject to false positives and false negatives. Accordingly, the development of more reliable detection and quantification methods, for example those based on polymerase chain reaction (PCR) assays20,21,24,is well justified. 
The ultimate goal for inoculation experiments should be to develop an efficient treatment that provides durable systemic resistance against herbivory and/or disease. A plausible, but as yet untested, hypothesis is that the extent of endophytic colonization should correlate positively with the extent of endophyte-mediated resistance. A natural next step after refining inoculation methods, therefore, could be to examine this correlation. Several video protocols can help researchers design a suitable resistance assay for a target pest or pathogen29-31. Ultimately, it is this assay what will determine the success of the inoculation method, and the associated potential for endophytic biological control.

Fungal entomopathogens are important regulators of insect populations with considerable potential as mycopesticides1. Only recently, however, have fungal entomopathogens been shown to occur as endophytes, both naturally and in response to various inoculation methods2. The ecological function of endophytic fungal entomopathogens remains largely unknown, but some studies have implicated them in plant growth3,4, herbivore resistance5-8, and disease resistance9,10. The overall objective of the methods presented here is to introduce a fungal entomopathogen as an endophyte, in preparation for subsequent evaluations of endophytic biological control. 
Beauveria bassiana (Balsamo) Vullemin (Ascomycota: Hypocreales) is the best-studied endophytic fungal entomopathogen5-9,11-19, and it is available as a commercial mycopesticide. Inoculation methods tested to establish B. bassiana as an endophyte include soil drenches14,17, seed coatings18 and immersions14, radicle dressings13,15, root and rhizome immersions11,16,18, stem injections17, foliar sprays14,17,20 and flower sprays19. Using these methods, researchers have introduced B. bassiana into banana11, bean7, cacao13, coffee17, corn7, cotton7, date palm12, jute21, opium poppy20, pumpkin7, radiata pine18, sorghum14, tomato7 and wheat7. Recent evidence suggests that endophytic B. bassiana has the potential to protect plants not only from arthropod pests5-7,22-27, but also from some plant pathogens9. 
The common bean (Phaseolus vulgaris) ranks among the crops most vulnerable to pests and diseases. It can be affected by more than 400 pests and 200 pathogens, whose attack is thought to be the most limiting bean production factor across regions28. Accordingly, the common bean may be an excellent model crop to examine the full spectrum of endophytic biological control by B. bassiana. As a first step in this direction, this article describes foliar sprays and soil drenches as inoculation methods to introduce B.bassiana as an endophyte in the common bean.

<table border="1">
<tbody>
	<tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 	<td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td></td>
 </tr>
 <tr>
 	<td>Mycotrol SE	</td>
 <td>Laverlam</td>
 <td>4167</td>
 <td></td>
 </tr>
 <tr>
 	<td>Noble agar</td>
 <td>Sigma</td>
 <td>A5431-250G</td>
 <td></td>
 </tr>
 <tr>
 	<td>Penicillin G sodium salt</td>
 <td>Sigma</td>
 <td>P3032-25MU</td>
 <td></td>
 </tr>
 <tr>
 	<td>Petri dish (100 x 15 mm)</td>
 <td>Fisher</td>
 <td>08-757-12</td>
 <td></td>
 </tr>
 <tr>
 	<td>Petri dish (60 x 15 mm)</td>
 <td>Fisher</td>
 <td>08-757-13A</td>
 <td></td>
 </tr>
 <tr>
 	<td>Potato dextrose agar</td>
 <td>Difco</td>
 <td>213400</td>
 <td></td>
 </tr>
 <tr>
 	<td>Regular bleach (NaOCl)</td>
 <td>CLOROX</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 	<td>Streptomycin sulfate salt</td>
 <td>Sigma</td>
 <td>S6501-25G</td>
 <td></td>
 </tr>
 <tr>
 	<td>Tetracycline</td>
 <td>Sigma</td>
 <td>T3258-25G</td>
 <td></td>
 </tr>
 <tr>
 	<td>Triple quince (NPK)</td>
 <td>ABOCOL</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 	<td>Triton X-100</td>
 <td>Sigma</td>
 <td>X-100</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 	<td>Biological safety cabinet</td>
 <td>NuAire</td>
 <td>NU-425-600</td>
 <td></td>
 </tr>
 <tr>
 	<td>Hemocytometer</td>
 <td>Fisher</td>
 <td>02-671-10 </td>
 <td></td>
 </tr>
 <tr>
 	<td>Leica DM LB microscope</td>
 <td>Leica</td>
 <td>N/A</td>
 <td></td>
 </tr>
 </tbody>
</table>

establishing fungal entomopathogens as endophytes: towards endophytic biological control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

B. bassiana was able to endophytically-colonize P. vulgaris in response to the demonstrated inoculation treatments (Figure 1). Both foliar sprays and soil drenches resulted in endophytic colonization by B. bassiana in over 80% of the treated plants (Figure 2). However, the extent of colonization depended on the plant part evaluated and the inoculation method used. Leaves responded best to spray inoculations. Roots, on the other hand, responded only to drench inoculations. Finally, stems responded similarly to both inoculation methods. B. bassiana was not detected in any of the control plant sections. 
Independent of the treatment, endophytes other than B. bassiana grew from 15% of the evaluated plant sections, but they were dissected out from media plates before they could invade neighboring sections and influence the results. 
Treatment and control plants were visibly indistinguishable two weeks after inoculations. No differences were detected in their dry weight and in their height. 
<img src="/files/ftp_upload/50360/50360fig1.jpg" alt="Figure 1"/> 
Figure 1. Representative results of inoculation treatments on endophytic colonization of bean plants (Phaseolus vulgaris cv. Calima) by Beauveria bassiana. Top left: control plates with no growth. Top right: Fungal endophyte from a plant section contaminating entire plate. Bottom left: B. bassiana growing from two plant sections. Bottom right: Endophytic B. bassiana conidia and conidiophores as seen under a microscope.
<img src="/files/ftp_upload/50360/50360fig2.jpg" alt="Figure 2" fo:content-width="4.5in" fo:src="/files/ftp_upload/50360/50360fig2highres.jpg"/> 
Figure 2. Effect of inoculation treatments on endophytic colonization of bean plants (Phaseolus vulgaris cv. Calima) by Beauveria bassiana, two weeks after inoculations with the strain GHA. Percent colonization represents the number of colonized plant sections divided by the number of cultured sections.

Watch this Scientific Journal Video about Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control at JoVE.com

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

establishing-fungal-entomopathogens-as-endophytes-towards-endophytic

Research

JoVE Journal

Bioengineering

34.4K Views.  International Center for Tropical Agriculture (CIAT), Cali, Colombia. This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

Video: Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

Experimental Methods to Study Human Postural Control

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied by appropriate mathematical methods are needed to understand the role of the different sub-systems involved in human postural control. This article describes a protocol for performing perturbed standing experiments, acquiring experimental data, and carrying out the subsequent mathematical analysis, with the aim of understanding the role of musculoskeletal system and central control in human upright posture. The results generated by these methods are important, because they provide insight into the healthy balance control, form the basis for understanding the etiology of impaired balance in patients and the elderly, and aid in the design of interventions to improve postural control and stability. These methods can be used to study the role of somatosensory system, intrinsic stiffness of ankle joint, and visual system in postural control, and may also be extended to investigate the role of vestibular system. The methods are to be used in the case of an ankle strategy, where the body moves primarily about the ankle joint and is considered a single-link inverted pendulum.

This article presents an experimental/analytic framework to study human postural control. The protocol provides step-by-step procedures for performing standing experiments, measuring body kinematics and kinetics signals, and analyzing the results to provide insight into the mechanisms underlying human postural control.

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...