This study has received funding from the Faculty of Dentistry, Mahidol University, Bangkok, Thailand. The authors thank Dr Erica Di Federico from School of Engineering and Materials Science and Dr Thomas Kelly from School of Geography at Queen Mary University of London for their expert technical inputs &#38; guidance in this work.

1. Tooth analog fabrication
<ol>
	<li>Anatomically reduce the occlusal surface of a typodont mandibular first molar (with bifurcated root) by 1 mm and bevel the margin using coarse and fine diamond burs.</li>
	<li>Scan the prepared typodont with a dental laboratory scanner.</li>
	<li>Open the scanned file with OrthoAnalyzer in the CAD software. On the Sculpt toolkit window, click on the Wax knife tool and set its diameter and level as 2.6 mm and 63&#160;&#956;m, respectively. Gradually pull each root surface toward each other to merge the bifurcated roots into a single root to facilitate the milling process (Figure 1).</li>
	<li>Mill the tooth analog dies from a high-pressure fiberglass laminate (n = 30) (Garolite, National Electrical Manufacturers Association [NEMA] grade G10) using a five-axis milling machine (Figure 2).</li>
</ol>
2. Mounting
<ol>
	<li>Use any appropriate CAD software (e.g., Autodesk Inventor Professional 2025) to design a jig that fits the root section of the model tooth and the space inside the PVC end caps to ensure standardized position and orientation of the test specimen.</li>
	<li>Print one jig per test toot in PMMA or similar modulus material using a 3D printer.</li>
	<li>Combine the root parts and dies with the PVC end cap, typically 25 mm in inner diameter, 21.5 mm high, and 5.5 mm in wall thickness.</li>
	<li>Mix the cold-curing low-viscosity epoxy resin and pour it up to the cementoenamel junction region of the model teeth. Be careful not to contaminate the occlusal surface of the model teeth with the flowing resin. Leave to fully set at room temperature for at least 24 h (Figure 3).</li>
</ol>
3. Occlusal veneer fabrication
<ol>
	<li>Import the scanned file of tooth analog to the CAD software.</li>
	<li>Under Directions, determine the insertion direction of the occlusal veneer.</li>
	<li>Under Interfaces, select Margin line and mark the margin line of the scanned tooth analog. Then, select Die interface | Advance settings and adjust the cement gap to 0.025 mm and the extra cement gap to 0.050 mm.</li>
	<li>Under Anatomy design, design a 1 mm-thick occlusal veneer using a template from Smile Library and adjust it with tools in Sculpt.</li>
	<li>Mill the lithium disilicate (IPS e.max CAD) and resin nanoceramic (Lava Ultimate) blocks (n=15 per group) using a five-axis milling machine and following the manufacturer&#39;s instructions (Figure 4).</li>
</ol>
4. Bonding and cementation
<ol>
	<li>Clean all master dies for 90 s in an&#160;ultrasonic machine and air dry. Then, apply 5% hydrofluoric acid to the occlusal surface for 60 s, thoroughly rinse with water, and air dry.</li>
	<li>For the LD group, etch the internal surface with 5% hydrofluoric acid for 20 s, thoroughly rinse with water, and air dry.</li>
	<li>For the RNC group, air-abrade with aluminum oxide powder grain size 50 &#181;m at 2 bars (200 kPa, 30 psi) for 10 s. Remove excess sand with alcohol and air dry.</li>
	<li>Apply silane, a universal bonding agent, and load dual-cured adhesive cement to the restoration intaglio. Place the restoration onto the prepared master dies by loading them under a silicone-filled compression head in an&#160;universal testing machine with a 40 N load.</li>
	<li>Cure with a light-emitting diode (LED) using light intensity in the normal mode of 1,000-1,200 mW/cm2 for 1-2 s. Remove excess cement, continue the curing light on each surface for 20 s, and leave it in the universal testing machine for 5 min.</li>
	<li>Remove from the universal testing machine and leave in distilled water at 37 &#176;C for 48 h to allow the cement to fully cure.</li>
	<li>Before testing, use fine permanent markers to draw three medial-lateral reference lines (sinistral, central, and dextral) and three anterior-posterior reference lines (upper, central, and lower) in different colors using fine permanent markers.</li>
</ol>
5. Quasistatic mechanical testing
<ol>
	<li>Position the test specimen in the center of the lower platen of a mechanical testing machine with a 5 kN load cell set up for compression testing.</li>
	<li>Place a 5.5 mm diameter stainless-steel ball in the central fossa of the restoration at the intersection of the central reference lines (Figure 5).</li>
	<li>Place a protective acrylic ring around the specimen and a debris shield in front of the testing machine to limit flying debris.</li>
	<li>Bring the crosshead down until nearly in contact with the steel ball and zero load and displacement.</li>
	<li>Apply compression at 1 mm/min until the restoration fractures, indicated by a sudden drop in the load. Record this load (Figure 5).</li>
	<li>After fracture, remove the shield and the acrylic ring, and carefully collect the test specimen and its fragments. The colored lines assist in placing the ceramic fragments into their original positions for subsequent analysis (Figure 5).</li>
	<li>Place the next test specimen and follow steps 5.2-5.6 until the groups are complete.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Fill in Minitab worksheets with material codes and maximum loading values (N) retrieved from the experiment in the first and second columns, respectively.</li>
	<li>Conduct an independent t-test for two samples by selecting Stat | Basic Statistics | 2-sample t. Set the confidence level at 95% and assume equal variances.</li>
	<li>Create the Weibull distribution plot by selecting Stat | Reliability/Survival | Distribution Analysis (Right Censoring) | Distribution ID Plot. Choose maximum loading values in the Variable box, tick and choose material names in the By variable box, specify distribution as Weibull, and click OK.</li>
	<li>Conduct the Weibull statistical analysis by selecting Stat | Reliability/Survival | Distribution Analysis (Right Censoring) | Parametric Distribution Analysis | Weibull | Graphs. Choose the Probability plot | Display confidence intervals on above plots and click OK.</li>
</ol>
7. Fractographic analysis
<ol>
	<li>For stereo microscopy, mount the eyepiece camera and capture images (20x) of the samples&#39; aerial and side views via the stereomicroscopy software.</li>
	<li>For scanning electron microscopy, cut the specimen to the cementoenamel junction (CEJ), put it in an acetone bath in an ultrasonic cleaner&#160;and then air dry. Gold-coat and capture images (250-300 magnification) of the samples&#39; aerial and side views.</li>
</ol>

Quasistatic Load-to-Fracture Test of CAD/CAM Occlusal Veneers on Dentin Analogs

The authors have no conflicts of interest to declare.

In recent years, minimally invasive occlusal veneers have increasingly received attention in contemporary restorative dentistry. These restorations are usually fabricated from monolithic CAD/CAM glass-matrix ceramic, polycrystalline, and hybrid materials26. Conservative tooth preparation, the ease of access and visibility during tooth preparation, impression taking and cementation, and preservation of the marginal gingiva have been promoted as advantages26,<sup c...

Metal-free restorations are now highly preferred in anterior and posterior dentition due to their excellent optical characteristics and biocompatibility1. However, the major drawback of such materials is their susceptibility to fracture2. Most ceramics are vulnerable to cracks generated by tensile stresses, even under low strain3. Fractures of dental ceramic prostheses usually develop from slow radial crack growth due to long-term exposure to the tensile stresses generated during chewing4. Their weaknesses escalate with intrinsic flaws or defects within the materials and extrinsic flaws from fabrication and postprocessing5. Flexural strength, the capability to withstand tensile stress, of dental CAD/CAM materials can be achieved and compared through standard tests such as uniaxial (3-point or 4-point bending) and biaxial bending tests (ball-on-ring, ring-on-ring, and piston-on-three balls). Meanwhile, fracture toughness, the capability of a material to resist crack growth, can be derived from a single-edged notch beam and an indentation test. However, these tests cannot entirely predict and represent the behavior of the cemented prostheses with different anatomical configurations6. Other monotonic or dynamic mechanical tests have been introduced to justify their performance with various clinical aspects7,8.
A load to fracture or &#34;crunch the crown&#34; test has been used extensively in dentistry to investigate and compare the strengths of ceramic restorations with complex geometries9,10. Monotonic uniaxial compression is quasistatically exerted on the restorations in a vertical or lateral direction until a catastrophic fracture occurs. The fracture strength of the material can be determined from the maximum loading force, whereas the modes of fracture, including the site and direction of the crack(s), can be examined microscopically. A good restoration should be able to withstand both compressive and tensile stress from the voluntary maximum bite force, the highest masticatory force generated by jaw-elevator muscles under the influence of craniomandibular biomechanics and the reflex pathway11,12, which could be up to 900 N in the posterior teeth3. Furthermore, bruxism can involuntarily increase the force to 1,200 N in the same region13. In addition to material properties (i.e., elastic modulus), geometries, thicknesses, adhesive cement, and defect distributions influence any prosthesis&#39;s strength14. However, arguments have been raised on the clinical relevance of such tests due to the nonclinical high forces and the failure mechanisms being dissimilar to clinical situations6,14. A fatigue resistance test involving step-stress analysis and intraoral condition may be a more realistic approach for predicting the longevity of dental restorations7. Nevertheless, the load to fracture is still a quick, simple, and repeatable in vitro test to compare the strengths of new CAD/CAM ceramics materials launched onto the market where the manufacturer&#39;s data may not be dependable15,16,17. The result may reflect the prosthesis&#39;s tolerance to extreme forces from parafunction activities and unexpected clinical situations such as biting on hard seeds or gravels, which also causes failure in dental prostheses18,19,20,21.
With their increasing use to rehabilitate posterior teeth, the mechanical performance of occlusal veneers made from milled and printed CAD/CAM materials has been investigated for various aspects, including types of materials, prosthetic designs, tooth abutment preparation design, thicknesses, surface treatments, adhesive bonding, and luting cement system22,23. However, the data are still limited, and the test materials are from glass matrix ceramic and conventional CAD/CAM composite materials. An alternative hybrid material, resin nanoceramic, is now available. It claims to incorporate the strength of nanoceramic fillers and resiliency from resin matrix, which may be suitable for thin, minimally invasive restoration. However, its mechanical performance, especially in the molar region, requires more supporting evidence for clinical implications.
Until now, researchers have not had materials that can substitute for natural teeth in laboratory testing. High-pressure fiberglass laminate (National Electrical Manufacturers Association; NEMA grade G10) with the tradename of Garolite has been proposed as a dentin analog material for the mechanical testing of dental ceramics since 201014. It is a thermoset composite material comprising multilayers of fiberglass soaked in epoxy resin under high pressure. It can withstand high-stress conditions with similar elastic properties, fatigue behavior, and adhesive bond strengths as hydrated dentin14,24. It provides advantages over natural teeth regarding specimen preparation, standardization, and ethical authorization, with time savings due to reduced biosafety concerns24. Surface treatment can be performed by etching with 5% or 10% hydrofluoric acid from 60 s to 90 s and applying a silane coupling agent14,24. Nevertheless, studies on cemented prostheses with this material are limited, and the reliability of the existing evidence is still questionable24,25.
In this study, a laboratory protocol for a monotonic load-to-fracture test of 1 mm-thick occlusal veneers cemented to the master dies milled from dentin analog material against a&#160;nonfixed stainless steel ball was developed. The maximum load-bearing capacities of two dental CAD/CAM materials: lithium disilicate (LD) - IPS e.max CAD and resin nanoceramic (RNC) - Lava Ultimate, with n = 15 per group, were quantified and statistically compared through a two-sample independent t-test and Weibull statistical analysis. The fracture patterns were also investigated under optical stereomicroscopy and scanning electron microscopy. The study hypothesis was that this was an appropriate method of modeling the failure of occlusal veneers in clinical applications. The statistical null hypothesis was that there should be no difference in the maximum load-bearing capacities between the occlusal veneers made from the two materials.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D printing (SLA)</td>
			<td>&#160;Formlabs, Somerville, MA, USA</td>
			<td></td>
			<td>Form3+</td>
		</tr>
		<tr>
			<td>3Shape Dental Designer CAD software&#160;</td>
			<td>3Shape A/S, Copenhagen, Denmark</td>
			<td></td>
			<td>CAD software for tooth analog and veneers</td>
		</tr>
		<tr>
			<td>5% hydrofluoric acid&#160;</td>
			<td>Ivoclar Vivadent, Schaan, Liechtenstein</td>
			<td></td>
			<td>IPS Ceramic Etching Gel</td>
		</tr>
		<tr>
			<td>Alumina powder</td>
			<td>Ronvig Dental Mfg. A/S, Daugaard, Denmark</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bluehill Universal materials testing software&#160;</td>
			<td>Instron Mechanical Testing Systems, Norwood, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CamLabLite software&#160;</td>
			<td>Bresser UK Ltd, Kent, UK</td>
			<td></td>
			<td>Stereomicroscopy Software</td>
		</tr>
		<tr>
			<td>Cold-curing low-viscosity epoxy resin&#160;</td>
			<td>Struers SAS, Champigny-sur-Marne, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-cure resin cement&#160;</td>
			<td>3M, Saint Paul, MN, USA</td>
			<td></td>
			<td>Rely X Ultimate Adhesive Resin Cement</td>
		</tr>
		<tr>
			<td>Eyepiece camera&#160;</td>
			<td>ToupTek Photonics Co., Ltd., Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-pressure fibreglass laminate discs&#160; (G10)</td>
			<td>PAR Group Ltd, Lancashire, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IPS e.max CAD</td>
			<td>Ivoclar Vivadent, Schaan, Liechtenstein</td>
			<td>YB54G9/605330</td>
			<td>Low translucency, A3, C14</td>
		</tr>
		<tr>
			<td>Laboratory scanner&#160;</td>
			<td>3Shape A/S, Copenhagen, Denmark</td>
			<td></td>
			<td>D900L</td>
		</tr>
		<tr>
			<td>Lava Ultimate</td>
			<td>3M ESPE, Saint Paul, MN, USA</td>
			<td>9541467/3314A3-LT</td>
			<td>Low translucency, A3, 14L</td>
		</tr>
		<tr>
			<td>Light-emitting diode (LED) curing light&#160;</td>
			<td>Woodpecker Medical Instrument, Guilin, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milling machine&#160;</td>
			<td>VHF camfacture AG, Amnnerbuch, Germany</td>
			<td></td>
			<td>VHF S2</td>
		</tr>
		<tr>
			<td>Minitab 18&#160;</td>
			<td>Minitab Inc, State College, PA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nQuery Advisor Version 9.2.10&#160;</td>
			<td>Statistical Solutions Ltd., CA, USA</td>
			<td></td>
			<td>Statistical Software</td>
		</tr>
		<tr>
			<td>Polyvinyl chloride end cap&#160;</td>
			<td>Plastic Pipe Shop Ltd, Stirling, UK</td>
			<td></td>
			<td>25 mm X 21.5 mm;</td>
		</tr>
		<tr>
			<td>Scanning electron microscope&#160;</td>
			<td>Tescan, Brno, Czech Republic</td>
			<td></td>
			<td>Tescan Vega</td>
		</tr>
		<tr>
			<td>Silane coupling agent&#160;</td>
			<td>3M, Saint Paul, MN, USA</td>
			<td></td>
			<td>RelyX Ceramic Primer</td>
		</tr>
		<tr>
			<td>Autodesk Inventor Professional 2024</td>
			<td>Autodesk, San Francisco, CA, USA</td>
			<td></td>
			<td>CAD software for jig</td>
		</tr>
		<tr>
			<td>Sputter vacuum coater&#160;&#160;</td>
			<td>Quorum, East Sussex, UK</td>
			<td></td>
			<td>MiniQS Sputter Coater</td>
		</tr>
		<tr>
			<td>Stata18&#160;</td>
			<td>StataCorp LLC, College Station, TX, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope&#160;</td>
			<td>Carl Zeiss AG, Oberkoche, Germany</td>
			<td></td>
			<td>Zeiss Stemi 508</td>
		</tr>
		<tr>
			<td>Typodont mandibular first molar&#160;</td>
			<td>Frasaco GmbH, Tettnang, Germany</td>
			<td></td>
			<td>ANA-4 Z3RN-36</td>
		</tr>
		<tr>
			<td>Universal dental bonding agent&#160;</td>
			<td>&#160;3M, Saint Paul, MN, USA</td>
			<td></td>
			<td>Scotch Bond Universal Adhesive</td>
		</tr>
		<tr>
			<td>Universal testing machine</td>
			<td>Instron Mechanical Testing Systems, Norwood, MA, USA</td>
			<td></td>
			<td>Intron 5900-84&#160;</td>
		</tr>
	</tbody>
</table>

quasistatic mechanical testing for computer-aided design and manufacturing occlusal veneers cemented to milled dentin analog material

Under current minimally invasive treatment regimes, minor tooth preparation and thinner biomimetic ceramic restoration are used to preserve the restored tooth&#39;s vitality, aesthetics, and function. New computer-aided design and computer-aided manufacturing (CAD/CAM) ceramic-like material are&#160;now available. To guarantee longevity, a dental clinician must know these&#160;newly launched product&#39;s mechanical strength compared to the relatively brittle glass-matrix ceramic. Furthermore, a tooth substitute has been promoted for laboratory investigation, especially after the pandemic, and more evidentiary support is required for its application.
This study developed a laboratory protocol for a monotonic load-to-fracture test to determine the fracture strength of 1 mm-thick CAD/CAM occlusal veneers. Master dies were milled from high-pressure fiberglass laminate, which has&#160;similar elastic modulus and bond strength as hydrated dentin. They were mounted into polyvinyl chloride (PVC) end caps with cold-curing epoxy resin. Occlusal veneers, also called tabletop restorations, were milled from lithium disilicate (LD) and resin nanoceramic blocks (RNC) and cemented to prepared master dies using dual-cured adhesive resin cement. They were&#160;allowed to cure fully by storing in distilled water for 48 h at 37 &#176;C.
All samples were then placed in a universal testing machine and loaded via a non-fixed 5.5 mm stainless-steel ball that allows lateral movement as would occur against the antagonist&#160;teeth. Compression was applied at a 1 mm/min rate, and the load-displacement graph was generated. The average maximum load-bearing capacity of restorations in the RNC group (3,212.80 &#177; 558.67 N) was significantly higher than in the LD group (2727.10 &#177; 472.41 N) (p &#60; 0.05). No debonding was found during the test.&#160;Both CAD/CAM materials may have a similar flaw distribution. Hertzian cone crack was found at the loading site, whereas radial cracks propagating from the cementation surface were found close to the margin in both groups.

The sample size calculation was performed using the referenced software, which generated an effect size of 0.39 and suggested a minimum sample size of n = 13 per group. However, a sample size of n = 15 was chosen in this study to detect the difference of 5%. The null hypothesis was rejected. Despite having greater flexural strength, the mean values of the maximum loading force of the 1 mm-thick occlusal veneers (n = 15 per group) made from the LD (lithium disilicate: 2,727.10 &#177; 472.41 N) group were significantly low...

Quasistatic Mechanical Testing for Computer-Aided Design and Manufacturing Occlusal Veneers Cemented to Milled Dentin Analog Material

A quasistatic load-to-fracture test with a non-fixed stainless steel ball was developed to determine the fracture strength of minimally invasive posterior computer-aided design and manufacturing restorations cemented to dentin analog materials. This test models the typical loading regime responsible for the fracture of dental restorations.

Under current minimally invasive treatment regimes, minor tooth preparation and thinner biomimetic ceramic restoration are used to preserve the restored ...

quasistatic-mechanical-testing-for-computer-aided-design

Research

JoVE Journal

Medicine

266 Views.  Queen Mary University of London. A quasistatic load-to-fracture test with a non-fixed stainless steel ball was developed to determine the fracture strength of minimally invasive posterior computer-aided design and manufacturing restorations cemented to dentin analog materials. This test models the typical loading regime responsible for the fracture of dental restorations.

Video: Quasistatic Mechanical Testing for Computer-Aided Design and Manufacturing Occlusal Veneers Cemented to Milled Dentin Analog Material

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular matrix (ECM), the protein meshwork that binds cells and tissues together. How the elastic properties of an ECM network relate to its composition, and whether the relaxation properties of the ECM play a physiological role, are questions that have yet to be fully addressed. Part of the challenge lies in the complex architecture of most ECM systems and the difficulty in isolating ECM components without compromising their structure. One exception is the zonule, an ECM system found in the eye of vertebrates. The zonule comprises fibers hundreds to thousands of micrometers in length that span the cell-free space between the lens and the eyewall. In this report, we describe a mechanical technique that takes advantage of the highly organized structure of the zonule to quantify its viscoelastic properties and to determine the contribution of individual protein components. The method involves dissection of a fixed eye to expose the lens and the zonule and employs a pull-up technique that stretches the zonular fibers equally while their tension is monitored. The technique is relatively inexpensive yet sensitive enough to detect alterations in viscoelastic properties of zonular fibers in mice lacking specific zonular proteins or with aging. Although the method presented here is designed primarily for studying ocular development and disease, it could also serve as an experimental model for exploring broader questions regarding the viscoelastic properties of elastic ECM&#39;s and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.

The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1.

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular ...