This study was supported by grants from the National Natural Science Foundation of China (81973493); National Interdisciplinary Innovation Team of Traditional Chinese Medicine (ZYYCXTD-D-202209); Sanajon Pharmaceutical Group Chengdu University of TCM production, study, and Research Joint Laboratory Project (2019-YF04-00086-JH); and Sichuan Province Science and Technology Plan Funded Project (2021YFN0100). The authors thank the Innovative Institute of Chinese Medicine and Pharmacy of Chengdu University of TCM for its technical support in the mass spectrometry work.

1. Extract preparation
<ol>
	<li>Accurately weigh an appropriate amount of PE, and add 10x (weight) of deionized water for reflux extraction.</li>
	<li>Set five samples for reflux extraction for 0 h (E1), 0.5 h (E2), 1 h (E3), 1.5 h (E4), and 2 h (E5) after weighing.</li>
	<li>After extraction, cool the samples to room temperature, and weigh to make up for the lost weight to ensure consistency with the pre-extraction weights.</li>
	<li>Centrifuge the samples at 8,581 &#215; g for 10 min to ensure the removal of insoluble material and herbal residues from the sample solution.</li>
	<li>Use a pipette to add 20 mL of sample solution into the sample bottle to ensure that the solution added each time is at the same height. 
	&#8203;NOTE: Avoid contamination, such as fingerprints, on the scanning part of the sample bottle, ensure that the sample bottle is clean, and check whether there are visible scratches on the bottle surface. When adding the sample solution, be careful not to spill or splash on the sample bottle, and ensure that the liquid level is at the same height in each bottle.</li>
</ol>
2. Instrument operation
<ol>
	<li>Turn on the MLS detection instrument, and warm it up for 30 min.</li>
	<li>Click on the Create file button in the top menu (or click on the File | New file function) to create a new test file.</li>
	<li>Click on the Show Turbiscan Lab Temperature button in the top menu to set the instrument target temperature to 25 &#176;C. 
	NOTE: The set temperature of the instrument must be higher than the room temperature; otherwise, the sample temperature will be affected by the room temperature.</li>
	<li>Click on Program Scan in the top menu to enter the setup analysis program. Add the program to the list, and in the taskbar, add 5 min as a cycle, scan for 48 h to the analysis sequence, and set the balance time to 20 min. Select this analysis program for all the subsequent measurements.</li>
	<li>Move the prepared sample bottle into the MLS detection system. After setting up the program, click on Start to start the measurement. 
	&#8203;NOTE: Be careful not to shake the glass bottle when loading the sample. The measurement can only be started after the sample temperature and setting temperature are balanced.</li>
</ol>
3. Multiple light scattering analysis program setting
<ol>
	<li>After the data collection, click on the calculation parameters list to set the optical parameters to calculate the stability index (SI), particle size, and particle migration speed.</li>
	<li>Set the optical parameters as follows: the continuous phase light transmission intensity (T0) as 99.99% (water), the dispersed phase refractive index (np) as 1.36, and the continuous phase refractive index (nf) as 1.33.</li>
</ol>

<ol>
	<li>Huang, H. -. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+on+the+approaches+of+diverse+sedimentations+in+polyphenol+solutions:+An+integrated+chain+of+evidence+based+on+the+physical+phase,+chemical+profile,+and+sediment+elements.">Exploration on the approaches of diverse sedimentations in polyphenol solutions: An integrated chain of evidence based on the physical phase, chemical profile, and sediment elements.</a> Frontiers in Pharmacology. 10, 1060 (2019).</li><li>Ran, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+or+low+temperature+extraction,+which+is+more+conducive+to+Triphala+against+chronic+pharyngitis.">High or low temperature extraction, which is more conducive to Triphala against chronic pharyngitis.</a> Biomedicine and Pharmacotherapy. 140, 111787 (2021).</li><li>Wei, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatoprotective+effects+of+different+extracts+from+Triphala+against+CCl(4)-induced+acute+liver+injury+in+mice.">Hepatoprotective effects of different extracts from Triphala against CCl(4)-induced acute liver injury in mice.</a> Frontiers in Pharmacology. 12, 664607 (2021).</li><li>Huang, H. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+stability+control+strategy+of+Triphala+solution+based+on+the+balance+of+physical+stability+and+chemical+stabilities.">Study on the stability control strategy of Triphala solution based on the balance of physical stability and chemical stabilities.</a> Journal of Pharmaceutical and Biomedical Analysis. 158, 247-256 (2018).</li><li>Bhattacharya, A., Chatterjee, A., Ghosal, S., Bhattacharya, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+activity+of+active+tannoid+principles+of+Emblica+officinalis+(amla).">Antioxidant activity of active tannoid principles of Emblica officinalis (amla).</a> Indian Journal of Experimental Biology. 37 (7), 676-680 (1999).</li><li>Chao, P. C., Hsu, C. C., Yin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+and+anti-coagulatory+activities+of+caffeic+acid+and+ellagic+acid+in+cardiac+tissue+of+diabetic+mice.">Anti-inflammatory and anti-coagulatory activities of caffeic acid and ellagic acid in cardiac tissue of diabetic mice.</a> Nutrition and Metabolism. 6, 33 (2009).</li><li>Tiwari, V., Kuhad, A., Chopra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emblica+officinalis+corrects+functional,+biochemical+and+molecular+deficits+in+experimental+diabetic+neuropathy+by+targeting+the+oxido-nitrosative+stress+mediated+inflammatory+cascade.">Emblica officinalis corrects functional, biochemical and molecular deficits in experimental diabetic neuropathy by targeting the oxido-nitrosative stress mediated inflammatory cascade.</a> Phytotherapy Research. 25 (10), 1527-1536 (2011).</li><li>Baliga, M. S., Dsouza, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amla+(Emblica+officinalis+Gaertn),+a+wonder+berry+in+the+treatment+and+prevention+of+cancer.">Amla (Emblica officinalis Gaertn), a wonder berry in the treatment and prevention of cancer.</a> European Journal of Cancer Prevention. 20 (3), 225-239 (2011).</li><li>Rehman, H. -. u., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+chemical+constituents+of+Phyllanthus+emblica.">Studies on the chemical constituents of Phyllanthus emblica.</a> Natural Product Research. 21 (9), 775-781 (2007).</li><li>Jang, Y., Koh, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+and+storage+stability+of+aronia+anthocyanins+encapsulated+with+combinations+of+maltodextrin+with+carboxymethyl+cellulose,+gum+Arabic,+and+xanthan+gum.">Characterisation and storage stability of aronia anthocyanins encapsulated with combinations of maltodextrin with carboxymethyl cellulose, gum Arabic, and xanthan gum.</a> Food Chemistry. 405, 135002 (2022).</li><li>Fu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+phenylalanine-modified+magnetic+ferroferric+oxide+nanoparticles+for+ciprofloxacin+removal+from+aqueous+solution).">Novel phenylalanine-modified magnetic ferroferric oxide nanoparticles for ciprofloxacin removal from aqueous solution).</a> Journal of Colloid and Interface Science. 632, 345-356 (2023).</li><li>Jiang, T., Charcosset, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+curcumin+within+oil-in-water+emulsions+prepared+by+premix+membrane+emulsification:+Impact+of+droplet+size+and+carrier+oil+on+the+chemical+stability+of+curcumin.">Encapsulation of curcumin within oil-in-water emulsions prepared by premix membrane emulsification: Impact of droplet size and carrier oil on the chemical stability of curcumin.</a> Food Research International. 157, 111475 (2022).</li></ol>

The authors have no conflicts of interest to disclose.

The rapid and accurate assessment of TCM stability has always been a focus of TCM research. To provide more useful information for directing the improvement of the extraction process, this study analyzed the stability and instability mechanisms of a sample using a near-infrared non-destructive technology.
In this protocol, the important stability parameters are calculated based on accurate MLS scan data. MLS scans can collect the transmission (T%) and backscattering (BS%) of ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65130">Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods</a>. The Authors section was updated from:
Haozhou Huang1 
Mengqi Li2 
Chuanhong Luo3 
Sanhu Fan4 
Taigang Mo4 
Li Han3 
Dingkun Zhang3 
Junzhi Lin5 
1Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine 
2Sichuan Nursing Vocational College 
3School of Pharmacy/School of Modern Chinese Medicine Industry, State Key Laboratory of Characteristic Chinese Medicine Resources in Southwest China 
4Sanajon Pharmaceutical Group 
5TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine
to
Haozhou Huang1,2 
Mengqi Li3 
Chuanhong Luo4 
Sanhu Fan5 
Taigang Mo5 
Li Han4 
Dingkun Zhang4 
Junzhi Lin6 
1State Key Laboratory of Southwestern Chinese Medicine Resources, Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine 
2Meishan Hospital of Chengdu University of Traditional Chinese Medicine 
3Sichuan Nursing Vocational College 
4State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine 
5Sanajon Pharmaceutical Group 
6TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65130">Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods</a>. The Authors section was updated.

Erratum: Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

In the manufacturing of traditional Chinese medicine (TCM), the stability of the TCM extraction intermediates and related liquid preparations has always been the focus of inspection1. The clinical efficacy of medicinal products, especially with polyphenols as the primary active ingredient, suffers due to significant stability issues2,3. Sanajon oral liquid and Nuodikang oral liquid are examples of typical cases of this issue4. Therefore, it is crucial to learn how to use efficient tools to rapidly and accurately evaluate and optimize the stability of liquid intermediates in the TCM production process. Phyllanthus emblica L. (PE), a widespread medicinal plant in Southeast Asia, is thought to have good antioxidant properties5, as well as anti-inflammatory6, antibacterial7, and antitumor actions8. During the thermal extraction procedure, the tannins in PE transform violently9. Under catalysis with high temperatures, these tannins hydrolyze quickly to produce molecules such as gallic acid and ellagic acid, which lead to instability or precipitation due to their poor solubility1. Current methods for evaluating TCM stability, such as accelerated testing or centrifugation, are usually cumbersome4, which limits the further development of pertinent preparation processes.
Based on the principle of multiple light scattering (MLS), we established a fast stability evaluation method for PEF extracts and analyzed the instability mechanism. MLS is a measurement method based on the scanning of near-infrared light sources. Any solution system change results in a change in the light intensity. The incident light is scattered when it is absorbed or penetrated by the particles of the sample. The system records the transmission light signal when it passes through the sample; if the light transmittance of the sample is poor, the system records the backscattering light signal. Compared with visual observation, this can save a lot of time1 and can quickly and accurately analyze the instability phenomenon in detail, thus providing more useful information for guiding the optimization of the extraction process.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adjustable electric heating jacket</td>
			<td>Beijing Kewei Yongxing Instrument Co., Ltd</td>
			<td>MH-1000&#160;</td>
			<td>www.keweiyq.com</td>
		</tr>
		<tr>
			<td>Analytical balance(1/10000)</td>
			<td>Sartorious, Germany</td>
			<td>BSA224S&#160;</td>
			<td>www.sartorius.com.cn</td>
		</tr>
		<tr>
			<td>CNC ultrasonic instrument</td>
			<td>Kunshan Ultrasonic Instrument Co., Ltd</td>
			<td>KQ-500DE</td>
			<td>www.ks-csyq.com</td>
		</tr>
		<tr>
			<td>GL-16 high-speed centrifuge</td>
			<td>&#160;Sichuan Shuke Instrument Co., Ltd</td>
			<td>18091403</td>
			<td>www.sklxj.com</td>
		</tr>
		<tr>
			<td>Phyllanthus emblica L.</td>
			<td>Hehuachi medicinal materials market</td>
			<td>&#160;YJL2004</td>
			<td>Produced in Yunnan</td>
		</tr>
		<tr>
			<td>Turbisoft Lab multiple light scattering instrument</td>
			<td>French Formulaction Company</td>
			<td>Turbisoft Lab 2.3.1.125 Fanalyser 1.3.5</td>
			<td>www.formulaction.com</td>
		</tr>
		<tr>
			<td>UPR-II-5T ultra-pure water device</td>
			<td>Sichuan ULUPURE&#160; Ultrapure Technology Co., Ltd</td>
			<td>Z16030559</td>
			<td>www.ccdup.com</td>
		</tr>
	</tbody>
</table>

using multiple light scattering to examine the stability of phyllanthus emblica l. extracts obtained with different extraction methods

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact on the effectiveness and quality of the final product. However, existing stability evaluation methods are often time-consuming and labor-intensive, requiring long-term observation and the operation of complex equipment (such as high-performance liquid chromatography), and it is difficult to obtain more physical information about the instability of the system. Therefore, there is an urgent need to establish a fast and accurate stability analysis technology for traditional Chinese medicine. Multiple light scattering is a cutting-edge analytical method that can accurately and rapidly evaluate the stability of traditional Chinese medicines in an environment-friendly manner without changing the nature or state of the sample or using organic reagents.
In this work, using the precise scanning data of multiple light scattering, the present protocol rapidly acquired the variation curves for layer thickness, particle migration speed, and average particle size over time. This enabled the precise identification of the mechanism and crucial characteristics causing the system&#39;s instability in its early stages. Of note, the research period for the extraction process can be considerably shortened by the detailed quantification of the system stability, which also allows for a quick, accurate, and in-depth analysis of the effects of various extraction processes on the stability of Phyllanthus emblica L.

Figure 1&#160;shows the principle of multiple light measurement and the meaning of the collected results. In the MLS spectra results (Figure 2), the abscissa was the height of the sample cell, and the ordinate was the transmission (T%) and backscattering (BS%) intensity. By calculating the MLS spectra results, the system can obtain the changes in the key physical parameters of the sample during the measurement period, including the delta transm...

Watch this Scientific Journal Video about Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods at JoVE.com

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

Here, we introduce a stability evaluation method based on multiple light scattering technology to evaluate the stability of traditional Chinese medicine extracts.

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact ...

using-multiple-light-scattering-to-examine-stability-phyllanthus

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561 Views.  Chengdu University of Traditional Chinese Medicine. Here, we introduce a stability evaluation method based on multiple light scattering technology to evaluate the stability of traditional Chinese medicine extracts.

Video: Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data

This report describes a novel Stata-based application of trigonometric regression modelling to 55 years of multiple sclerosis relapse data from 46 clinical centers across 20 countries located in both hemispheres. Central to the success of this method was the strategic use of plot analysis to guide and corroborate the statistical regression modelling. Initial plot analysis was necessary for establishing realistic hypotheses regarding the presence and structural form of seasonal and latitudinal influences on relapse probability and then testing the performance of the resultant models. Trigonometric regression was then necessary to quantify these relationships, adjust for important confounders and provide a measure of certainty as to how plausible these associations were. Synchronization of graphing techniques with regression modelling permitted a systematic refinement of models until best-fit convergence was achieved, enabling novel inferences to be made regarding the independent influence of both season and latitude in predicting relapse onset timing in MS. These methods have the potential for application across other complex disease and epidemiological phenomena suspected or known to vary systematically with season and/or geographic location.

Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude.

This report describes a novel Stata-based application of trigonometric regression modelling to  55 years of multiple sclerosis relapse data from 46 ...

Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can improve strength in individuals with MS, however; it remains unclear the duration of RST that is needed to make strength gains and how to adapt hip strengthening exercises for individuals of varying strength using only resistance bands. This paper describes the methodology to set up and implement an adapted resistance strength training program, using resistance bands, for individuals with MS. Directions for pre- and post-strength tests to evaluate efficacy of the strength-training program are included. Safety features and detailed instructions outline the weekly program content and progression. Current evidence is presented showing that significant strength gains can be made within 8 weeks of starting a RST program. Evidence is also presented showing that resistance strength training can be successfully adapted for individuals with MS of varying strength with little equipment.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis. Isolated muscle strengthening is a useful method to target specific weaknesses. This protocol describes a progressive resistance-training program using exercise bands to increase hip muscle strength.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

Muscle Function Obtained with Motion Mode Ultrasound and Surface Electromyography during Core Endurance Exercise

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured between fascial borders at a given time point during an exercise. This selected time point produces a one-dimensional image resulting in real-time, live observation of anatomy. Ultrasound used during functional movement can be referred to as dynamic ultrasound; this is feasible and reliable with the use of a linear transducer, elastic belt, and foam block to secure consistent transducer placement. The lateral abdominal wall is commonly investigated using ultrasound due to the overlapping nature of the muscles. Surface electromyography (sEMG) can complement M-mode ultrasound imaging because it measures the electrical representation of muscle activation. There is minimal evidence using M-mode ultrasound and sEMG simultaneously during core exercise. Exercises that challenge the core musculature involve both isometric holds (e.g., side plank), as well as oscillatory extremity movements (e.g., dead bug). In this study, both instruments will be used simultaneously to measure core muscle function during exercise. Ultrasound measurements will be obtained using a linear transducer and ultrasound unit, and sEMG measurements will be acquired from a wireless sEMG system. To make comparisons between participants and exercises, normalization methods using static, exercise starting positions for both instruments will be used. An activation ratio will be used for ultrasound and calculated by dividing the contracted thickness (thickness during a time point of exercise) by the rested (starting position) thickness. Muscle thickness will be measured in centimeters from the superior inferior fascial border to inferior superior fascial border. These methods aim to offer an innovative and practical measurement of muscle function with M-mode ultrasound and sEMG during core endurance exercises.

This protocol uses motion mode ultrasound and surface electromyography simultaneously to measure muscle function of the core. Muscle thickness and activation of the local stabilizers (e.g., transverse abdominis, internal oblique) and global movers (e.g., external oblique) is achievable during specific time points of the side plank and dead bug exercises.

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured ...

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally heterogeneous nanoparticle complexes, which exhibit varying sensitivity to the conditions required for the methodologies available to physicochemically characterize these agents. Currently, the critical quality attributes of iron-carbohydrate complexes have not been fully established. Dynamic light scattering (DLS) has emerged as a fundamental method to determine intact particle size and distribution. However, challenges still remain regarding the standardization of methodologies across laboratories, specific modifications required for individual iron-carbohydrate products, and how the size distribution can be best described. Importantly, the diluent and serial dilutions used must be standardized. The wide variance in approaches for sample preparation and data reporting limit the use of DLS for the comparison of iron-carbohydrate agents. Herein, we detail a robust and easily reproducible protocol to measure the size and size distribution of the iron-carbohydrate complex, iron sucrose, using the Z-average and polydispersity index.

Dynamic light scattering (DLS) has emerged as a suitable assay for evaluating the particle size and distribution of intravenously administered iron-carbohydrate complexes. However, the protocols lack standardization and need to be modified for each iron-carbohydrate complex analyzed. The present protocol describes the application and special considerations for the analysis of iron sucrose.

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...