This research was supported by the National Natural Science Foundation of China (Grant No. 82074059), the Open Fund for Key Laboratory of Xin&#39;an Medical Ministry of Education, Anhui University of Traditional Chinese Medicine (No.2022XAYX07), the Anhui Provincial Key Laboratory of Traditional Chinese Medicine Compound Open Fund funded project (No.2019AKLCMF03), the Anhui Province Academic Leader Reserve Candidate Funding Project (No. 2022H287), and the Anhui Provincial Health Research Key Project (AHWJ2022a013)

1. Preparation of the Taohong extract
<ol>
	<li>Measure 39.6 g of prepared rehmannia root (Rehmannia glutinosa [Gaetn.] Libosch. ex Fisch. et Mey.), 29.7 g of peach kernel (Prunus persica [L.] Batsch), 19.8 g of safflower (Carthamus tinctorius L.), 29.7 g of angelica (Angelica sinensis [Oliv.] Diels), 29.7 g of white peony root (Cynanchum otophyllum Schneid), and 19.8 g of ligusticum chuanxiong (Ligusticum chuanxiong hort), totaling 168.3 g of medicinal materials11, and place these ingredients in a decoction casserole.</li>
	<li>Perform decoction three times.
	<ol>
		<li>For each round, add 1,683 mL of water, and boil the ingredients for 1.5 h.</li>
	</ol>
	</li>
	<li>Combine the filtrate, and filter the resultant liquid with gauze. Concentrate the liquid in a rotary evaporator to a final volume of 400 mL.</li>
	<li>Evenly pour the concentrate into an evaporation dish, and place in a water bath at 90 &#176;C to dry the excess water.</li>
	<li>Place the thickened extract in a vacuum drying oven at 85 &#176;C, and dry for 8 h to obtain a dry extract.</li>
	<li>Subsequently, grind the dried extract into powder in a mortar, and then sieve with an 80-mesh sieve. 
	&#8203;NOTE: The resultant dry extract was in the form of a pink herbal powder containing the four medicinal ingredients.</li>
</ol>
2. Screening of the fillers
<ol>
	<li>Use lactose, pre-gelatinized starch, and microcrystalline cellulose as the fillers.</li>
	<li>Use cross-linked polyvinyl pyrrolidone (PVPP) as a disintegrant and magnesium stearate as a lubricant. 
	NOTE: Three formulas were used to generate prescriptions for testing.</li>
	<li>Prepare Formula 1: 0.5 g of medicinal powder and 3.4 g of microcrystalline cellulose (MCC) to produce the filler and 1 g of PVPP and 0.1 g of magnesium stearate to produce the lubricant.</li>
	<li>Prepare Formula 2: 0.5 g of medicinal powder, 3.4 g of pre-gelatinized starch as the filler, 1 g of PVPP as the disintegrant, and 0.1 g of magnesium stearate as the lubricant.</li>
	<li>Prepare Formula 3: 0.5 g of medicinal powder, 3.4 g of lactose as the filler, 1 g of PVPP as the disintegrant, and 0.1 g of magnesium stearate as the lubricant.</li>
	<li>Mix each prescription thoroughly, and pass through an 80-mesh screen. Place the evenly mixed powder into a single punch tablet machine, and punch out a tablet.</li>
	<li>Test each tablet separately. Place the tablet into a beaker with 1 L of 37 &#176;C distilled water.</li>
	<li>Place the beaker into a dissolution tester (Table of Materials).</li>
	<li>Start the dissolution tester, and time the reaction. When the tablets are completely disintegrated, stop the timer, and observe whether the tablets are evenly dispersed. 
	&#8203;NOTE: We considered the disintegration time and dispersion uniformity as indicators in the subsequent experiments. The data arising from each prescription are given in Table 1.</li>
</ol>
3. Disintegrant selection
<ol>
	<li>Mix the three excipients in pairs to create three different prescriptions, and test the combinations.</li>
	<li>Prepare formulation 1 with PVPP and low-substituted hydroxypropyl cellulose (L-HPC) (1:1).</li>
	<li>Prepare formulation 2 with L-HPC and carboxymethyl starch sodium (CMS-NA) (1:1).</li>
	<li>Prepare formulation 3 with PVPP and CMS-Na (1:1).</li>
	<li>Mix each prescription thoroughly, and pass through an 80-mesh screen. Place the evenly mixed powder into a single punch tablet machine.</li>
	<li>Investigate the appearance and shape of the tablets. Place the tablets on a white background, and observe whether the appearance of the color is uniform. Measure the disintegration time as per steps 2.7-2.9. 
	&#8203;NOTE: The results are shown in Table 2.</li>
</ol>
4. Optimization of the filler and disintegrant dosage
<ol>
	<li>Optimize the specific dosage of the fillers (step 2) and disintegrants (step 4) by performing an orthogonal experiment using the dosage of microcrystalline cellulose as factor A, the dosage of crosslinked polyvinylpyrrolidone as factor B, the dosage of sodium carboxymethyl cellulose as factor C, and the blank error as factor D.</li>
	<li>Prepare the excipients according to Table 3. Weigh the excipients separately, and mix the three formulations evenly.</li>
	<li>Pass the formulations through a sieve with a screen diameter of 0.18 mm.</li>
	<li>Press the formulations into tablets. Measure the disintegration time as per steps 2.7-2.9. 
	NOTE: Table 3 and Table 4 show the factor-level arrangement and orthogonal experiment arrangement.</li>
	<li>Import the data obtained into spreadsheet software for data analysis. 
	&#8203;NOTE: Table 5 shows the results of the variance analysis, and Figure 1 shows the trends for each test index.</li>
</ol>
5. Screening of the drug loading
NOTE: Following the filler and disintegrant tests, the optimal contents of microcrystalline cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, and magnesium stearate were determined to be 4.4 g, 1.8 g, 1.2 g, and 0.1 g, respectively.
<ol>
	<li>While keeping the relative content of the filler, disintegrant, and lubricant constant, prepare prescription 1 with 1 g of medicinal powder, prescription 2 with 1.5 g of medicinal powder, prescription 3 with 2 g of medicinal powder, and prescription 4 with 2.5 g of medicinal powder.</li>
	<li>Thoroughly mix the powder, filling agent, disintegrant, and lubricant used in each prescription, and pass through an 80-mesh sieve.</li>
	<li>Press the tablets, and place a tablet in the disintegration time tester.</li>
	<li>Start the instrument and time; stop the timer after the tablet has completely disintegrated. 
	&#8203;NOTE: The prescription schedule is shown in Table 6.</li>
</ol>
6. Quality evaluation of the Taohong Siwu dispersible tablets
<ol>
	<li>Appearance evaluation 
	NOTE: According to the requirements of the 2020 edition of the Chinese Pharmacopoeia General Rule 010112, the appearance of the tablet should be complete and smooth, and the color should be uniform.
	<ol>
		<li>Randomly select six pieces from each of the three batches to observe whether the surface of the dispersible sheet is smooth and the color is uniform.</li>
	</ol>
	</li>
	<li>Weight variation
	<ol>
		<li>For the evaluation of the wet weight, take 20 tablets from each batch, and accurately determine the total weight. Determine the mean tablet weight.</li>
		<li>Weigh each tablet separately.</li>
		<li>Compare the weight of each tablet with the mean weight. 
		NOTE: The difference between the mean weight and the weight of an individual tablet should not exceed 0.30 g &#177; 7.5%. The difference in weight should not exceed that of two tablets, and the weight of no single tablet should exceed the limit by a factor of one.</li>
	</ol>
	</li>
	<li>Disintegration and dispersion uniformity tests 
	NOTE: According to the provisions of General Rule 0921 of the Chinese Pharmacopoeia12, we determined the disintegration time for each batch of tablets.
	<ol>
		<li>Hang a gondola on a bracket with the stainless-steel shaft at the upper end. Immerse the apparatus in a 1 L beaker, and adjust the position of the gondola so that the screen is 25 mm from the bottom of the beaker when it drops to its lowest point.</li>
		<li>Fill the beaker with water at a temperature of 37 &#176;C &#177; 1 &#176;C, and adjust the height of the water level such that the sieve is 15 mm below the water surface when the gondola rises to its highest point. Ensure that the top of the gondola is not submerged in solution at any point.</li>
		<li>Take six tablets from each test batch, and place them in a glass tube in the gondola.</li>
		<li>Activate the dissolution tester. 
		NOTE: Each tablet needs to be completely disintegrated within 15 min.</li>
		<li>If one tablet is not completely disintegrated, then take another six tablets for re-testing. All the tablets must meet the required regulations.</li>
	</ol>
	</li>
</ol>
7. Characterization
<ol>
	<li>Chromatographic conditions
	<ol>
		<li>Use a chromatographic column with a column of 2.1 mm x 100 mm (e.g., ACQUITY UPLC BEH C18 1.7 &#181;m).</li>
		<li>Use a mobile phase of 100% acetonitrile (A) and 0.5% phosphoric acid aqueous solution (B).</li>
		<li>For gradient elution, use the following elution program: 0-4 min (10%-30%), 4-10 min (30%-60%), 10-15 min (60%-85%), 15-17 min (85%-50%) %), 17-20 min (50%-110%), 20-22 min (10%-10%).</li>
		<li>Use a flow rate of 0.2 mL/min, a detection wavelength of 260 nm, a column temperature of 30 &#176;C, and an injection volume of 2 &#181;L.</li>
	</ol>
	</li>
	<li>Solution preparation
	<ol>
		<li>Prepare an amygdalin reference solution. Dissolve the amygdalin in methanol at a concentration of 0.2 mg&#183;mL&#8722;1.</li>
		<li>Prepare a test solution.
		<ol>
			<li>For each sample, crush five tablets with a mortar, and place in a 25 mL volumetric flask with 25 mL of 80% methanol, volumetric to scale.</li>
			<li>Ultrasonicate each sample at 150 W and 40 kHz for 30 min to dissolve.</li>
			<li>Cool each sample to room temperature, and then add 80% methanol to a total volume of 25 mL.</li>
			<li>Finally, filter the sample with a microporous membrane (0.22 &#181;m). Use the substrate as the product for testing.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Linear relationship analysis
	<ol>
		<li>Take 1 mL of the reference solution, and filter with a microporous filter membrane (0.22 &#181;m).</li>
		<li>Transfer 0.5 &#181;L, 1 &#181;L, 1.5 &#181;L, 2 &#181;L, 2.5 &#181;L, and 3 &#181;L of the reference solution into the chromatography column, and perform chromatography as described in section 7.1.</li>
		<li>Inject the sample, and record the peak area.</li>
		<li>Perform linear regression using the peak area (y) and injection volume (x) as the variables. 
		NOTE: The regression equation obtained was y = 18115x &#8722; 2386.6 (R&#178; = 0.9993), thus indicating that amygdalin exhibits a good linear relationship in the range of 0.0492-0.3101 mg. Figure 2 shows the standard curve.</li>
	</ol>
	</li>
	<li>Precision testing
	<ol>
		<li>Inject 2 &#181;L of the reference solution (prepared as described in section 5.2.1) into the chromatograph six times successively.</li>
		<li>Record the peak area, and calculate the relative standard deviation (RSD) value. 
		NOTE: The measured RSD value for the peak area of amygdalin was 2.7%, thus indicating that the instrument exhibited good precision.</li>
	</ol>
	</li>
	<li>Repeatability experiments
	<ol>
		<li>To evaluate the repeatability, grind 30 dispersible tablets into a fine powder, and mix the resultant powders together.</li>
		<li>Divide the pooled powder into six batches weighing approximately 1 g each.</li>
		<li>Prepare the test solution as described in section 7.2.2.</li>
		<li>Inject 2 &#181;L of each sample, record the peak area, and calculate the RSD value. 
		NOTE: The measured peak area RSD value was 1.8%, thus indicating that the method exhibited good repeatability.</li>
	</ol>
	</li>
	<li>Stability experiments
	<ol>
		<li>Using the same test solution, inject 2 &#181;L of solution at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 14 h.</li>
		<li>Record the peak area, and calculate the RSD value. 
		NOTE: The measured RSD value of the peak area of amygdalin was 2.8%, thus indicating that the test solution was stable for 24 h at room temperature.</li>
	</ol>
	</li>
	<li>Sample recovery testing
	<ol>
		<li>Take six samples of dispersible tablets, and prepare test solutions according to the method described in section 7.2.2.</li>
		<li>Add 1 mL of the control substance to the same injection bottle, measure the sample content according to the conditions described in section 7.1, and calculate the recovery rate. 
		NOTE: The analysis showed that the mean recovery rate of amygdalin (n = 6) was 101% and that the RSD value was 2.8%, thus indicating that the method exhibited good accuracy.</li>
	</ol>
	</li>
	<li>Determination of the sample content
	<ol>
		<li>Use three batches of dispersible tablets to prepare test solutions in accordance with the method given in section 7.2.2.</li>
		<li>Inject 2 &#181;L of each sample under the chromatographic conditions described in section 7.1.</li>
		<li>Record the peak area, and measure the content. 
		NOTE: The mean content of amygdalin in the three batches of dispersible tablets was 0.257 mg/tablet.</li>
	</ol>
	</li>
	<li>Dissolution determination
	<ol>
		<li>Take six batches of dispersible tablets, and measure the dissolution rate according to the small cup method for dissolution determination described in the 2020 edition of Chinese Pharmacopoeia12.</li>
		<li>Use 0.1 M of hydrochloric acid (250 mL) as the dissolution medium, and perform the testing at 37 &#176;C &#177; 0.5 &#176;C and 100 rpm for 50 min.</li>
		<li>Immediately pass the samples through a microporous membrane (0.22 &#181;m).</li>
		<li>Inject and test according to the chromatographic conditions described in section 7.1.</li>
		<li>Record the peak area and dissolution rate. 
		NOTE: The dissolution rates of the six batches of samples were 98%, 99%, 96%, 97%, 97%, and 98%, respectively.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

In this study, we tested the effect of filler, disintegrant, and medicinal powder dosage on the disintegration time and dispersion uniformity of dispersible tablets using an orthogonal design. We found that the preferred formulation disintegrated rapidly. When selecting the most appropriate filler, we found that although lactose exhibited the shortest disintegration time, the hardness of these tablets was not sufficient. Furthermore, the surface of the tablets was not smooth enough, and there was evidence of powder loss and loose tablets. As such, lactose did not meet the requirements of an appropriate filler; therefore, we selected microcrystalline cellulose as the optimal filler. Microcrystalline cellulose is a polymer in the form of powder or short rods with strong fluidity and without a fibrous structure13. Moreover, microcrystalline cellulose is odorless, non-toxic, easy to disintegrate, and non-reactive with drugs. This polymer is an important excipient in the pharmaceutical industry and can efficiently bond drug components to promote drug molding14. Furthermore, this polymer can facilitate the decomposition of drug components while enhancing drug strength and is used mainly as an excipient, filler, or drug release modifier for the preparation of drug tablets, drug granules, and drug capsules15,16.
Disintegrants have good water absorption and swelling properties and can promote the uniform dispersion of dispersible tablets in water. At present, the most commonly used disintegrants in dispersible tablets are L-HPC, cross-linked PVPP, and CMS-Na17. In this experiment, the combination of cross-linked PVPP and CMS-Na exhibited the shortest disintegration time. Most dispersible tablets use two or more disintegrants. Combinations of disintegrants can improve the disintegration effect and reduce costs18. When screening drug loading, we found that as the drug load increased, the hardness of the tablets decreased; this effect may be related to the nature of the medicinal material powder19. Finally, the process used to prepare the dispersible tablets (containing four ingredients) was optimized by formulation. This was followed by the indexing of the disintegration time and dispersion uniformity. The final ingredients were medicinal powder (17%), microcrystalline cellulose (49%), cross-linked polyvinylpyrrolidone (20%), sodium carboxymethyl starch (13%), and magnesium stearate accounting (1%).
Compared with traditional Chinese medicine decoctions, dispersible tablets can exert good therapeutic effects with high bioavailability, good stability, and easy portability, especially for patients who find it difficult to take decoctions and experience difficulties with swallowing20,21. In traditional Chinese medicine, the main drug in dispersible tablets is not usually a single compound; rather, the drug is composed of a complex mixture of ingredients. Furthermore, such powders have a relatively high viscosity and are usually able to absorb moisture. Ordinary tablets containing Chinese herbal medicine powder as the main drug are associated with a wide variety of problems, including a long disintegration time and poor dispersion uniformity, which influence the curative effect. Therefore, in the present research, we designed a new formulation, dispersible tablets, for the Taohong Siwu decoction to solve the problems associated with traditional decoctions, thus expanding the range of applications and promoting absorption in the body22. In this research, we extracted a dry powder from medicinal materials by applying the water extraction method. Traditional decoction methods involve complex ingredients that can be extracted according to the effective components and the characteristics of the active ingredients. Furthermore, the content of the active ingredients can be increased in dispersible tablets. The separation of impurities in medicinal materials and the preservation of their active ingredients is a problem that should be considered carefully during preparation. It is also important to provide favorable conditions for the preparation of subsequent dispersible tablets. Traditional decoctions such as the Taohong Siwu decoction generally have a high sugar content. Herbal powders can be readily prepared so that they absorb moisture. Therefore, it is important to take precautions when drying during the preparation. Furthermore, the finished tablets should be resistant to moisture. 
It should be noted that this study only investigated one index component in the content determination experiments; this represents a notable limitation to this study, although our work provides a key foundation for subsequent in-depth research. Transforming a decoction of Taohong Siwuinto a dispersible tablet that is convenient for patients, more practical, and more feasible is also in line with the developing trends in traditional Chinese medicine preparations.

Taohong Siwu decoction is a prescription composed of peach kernel, safflower, angelica, white peony, ligusticum chuanxiong, and rehmannia1,2 that can be used to promote blood flow and reduce detumescence and pain3,4. Previous research has found that Taohong Siwu Tang is difficult to preserve and easily affected by mildew and other factors. Furthermore, the commonly used clinical decoction is inconvenient to transport, while the decoction procedure is laborious and subject to variations in quality. Therefore, there is an urgent need to develop a new formulation for Taohong Siwu tablets. 
Dispersible tablets are those that can be rapidly disintegrated in water and distributed in uniform suspensions5. In traditional Chinese medicine (TCM), the main drug in dispersible tablets is normally prepared from the raw materials of the TCM powder, extracts, and other appropriate excipients6. Compared with conventional liquid preparations and ordinary tablets, dispersible tablets, as a new dosage form, are more rapidly absorbed in the human body, exhibit better stability, are easy to take and transport, and exhibit a series of beneficial characteristics, such as rapid disintegration, high bioavailability, and good dispersion uniformity7,8.
The orthogonal test design method utilizes an orthogonal table to scientifically select the test conditions, arrange a reasonable test plan, and then use the mathematical concepts of range and variance to analyze the test results and generate an optimal plan9,10. In this study, we are particularly interested in certain key characteristics: the uniformity of dispersion, neatness, and comparability. Orthogonal experimental designs can also be adapted to incorporate a fractional and factorial design. In the present study, we use an orthogonal experimental design to screen and optimize the excipient types, excipient ratios, and drug loading data for dispersible Taohong Siwu tablets. Compared with traditional decoctions of Chinese medicine, the dispersible Taohong Siwu tablets prepared by the methodology described herein are easy to take, easy to preserve, and highly resistant to mildew.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>OCEANPAK</td>
			<td>A22T0218</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxymethyl starch sodium</td>
			<td>Maclean</td>
			<td>C12976293</td>
			<td></td>
		</tr>
		<tr>
			<td>Crosslinked Polyvinylpyrrolidone</td>
			<td>Maclean</td>
			<td>C12976293</td>
			<td></td>
		</tr>
		<tr>
			<td>Disintegration time limit tester</td>
			<td>Tianjin Guoming Pharmaceutical Equipment Co., Ltd.</td>
			<td>BJ-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric heating constant temperature drying oven</td>
			<td>Shanghai Sanfa Scientific Instrument Co., Ltd.</td>
			<td>DHG-9202&#183;2</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Electric thermostatic water bath&#160;</td>
			<td>Shanghai Sanfa Scientific Instrument Co., Ltd.</td>
			<td>DK-S24</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Balance</td>
			<td>Sartorius Scientific Instruments (Beijing) Co., Ltd.</td>
			<td>SQP</td>
			<td></td>
		</tr>
		<tr>
			<td>Intelligent Dissolution Tester</td>
			<td>Tianda Tianfa Technology Co., Ltd.</td>
			<td>ZRS-8L</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactose</td>
			<td>Maclean</td>
			<td>C12942141</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-Substituted Hydroxypropyl Cellulose</td>
			<td>Anhui Shanhe Pharmaceutical Excipients Co., Ltd.</td>
			<td>190219</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium stearate</td>
			<td>Maclean</td>
			<td>C12894996</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Alcohol</td>
			<td>TEDIA High Purity Solvents</td>
			<td>22075365</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcrystalline cellulose</td>
			<td>Maclean</td>
			<td>13028716</td>
			<td></td>
		</tr>
		<tr>
			<td>Single punch tablet machine</td>
			<td>Nantong Shengkaia Machinery Co., Ltd.</td>
			<td>TDP-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tablet hardness tester</td>
			<td>Shanghai Huanghai Drug Testing Instrument Co., Ltd.</td>
			<td>YPJ-200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Taohong Siwu Soup Extract</td>
			<td>self made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Taoren, Honghua,&#160; etc. traditional Chinese medicine&#160;</td>
			<td>The First Affiliated Hospital of Anhui University of Chinese Medicine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Acquity H-Class Ultra High Liquid Chromatography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

formation of dispersible taohong siwu tablets

Here, we optimize the process used to formulate and prepare dispersible Taohong Siwu tablets and provide a foundation for expanding their clinical application. Taking dispersion uniformity and disintegration time as the indices for investigation, we used a single-factor test to match and filter the excipient categories for Taohong Siwu tablets. The formulation was optimized by an orthogonal test design. The content and dissolution rates of the effective substances in dispersible Taohong Siwu tablets when prepared with optimized prescriptions were determined by ultra-high performance liquid chromatography (UPLC), and the optimal preparation process was determined.
The optimal composition for dispersible Taohong Siwu tablets was 17% Taohong Siwu extract powder, 1% magnesium stearate, 49% microcrystalline cellulose, 20% cross-linked polyvinylpyrrolidone, and 13% sodium carboxymethyl starch. When dispersible Taohong Siwu tablets were prepared by direct compression and the optimized prescription powder was uniformly dispersed within 3 min, the dissolution rate reached more than 90% within 50 min.When prepared according to the optimized methods,dispersible Taohong Siwu tablets disintegrate rapidly in water with good dispersion uniformity and controllable quality.

In this study, we used a traditional method to prepare liquid extracts of Taohong Siwu decoction and prepared an herbal powder by concentration and drying. By keeping the herbal powder, disintegrant, and lubricant unchanged, we screened r-lactose, pregelatinized starch, and microcrystalline cellulose as fillers to determine the disintegration time. Although the disintegration time with lactose as the disintegrant was better than that with pregelatinized starch and microcrystalline cellulose, the hardness and surface finish did not meet the required standards; thus, microcrystalline cellulose was selected as the filler.
To select an appropriate disintegrant, we established three prescriptions with the same herbal powder, filler, and lubricant: prescription 1 (cross-linked polyvinylpyrrolidone and low-substituted hydroxypropyl cellulose), prescription 2 (low-substituted hydroxypropyl cellulose and sodium carboxymethyl starch), and prescription 3 (crosslinked polyvinylpyrrolidone and sodium carboxymethyl starch); the ratio was 1:1 in each case. Taking appearance, disintegration time, and dispersion uniformity as the inspection indicators, we found the combined disintegration time and dispersion uniformity of crosslinked polyvinylpyrrolidone and sodium was optimal. Next, we performed orthogonal testing to identify the optimal filler and disintegrant content. The best results were obtained with 4.4 g of microcrystalline cellulose (MCC, Factor A), 1.8 g of crosslinked polyvinylpyrrolidone (PVPP, Factor B), and 1.2 g of sodium carboxymethyl starch (CMS-Na, Factor C).&#160;Once we had identified the optimal dosage and proportions of filler and disintegrant, we next identified the optimal amount of herbal powder by investigating the disintegration time, dispersion uniformity, and hardness of different proportions. Figure 3 shows that the four prescriptions passed the dispersion uniformity assessment. However, with a larger drug load, we observed a longer disintegration time and a reduction in harness. The final prescription was identified as 1.5 g of medicinal powder, 4.4 g of microcrystalline cellulose, 1.8 g of cross-linked polyvinylpyrrolidone, 1.2 g of sodium carboxymethyl starch, and 0.1 g of magnesium stearate. The final specification was 0.30 g per tablet and four tablets per administration (three times per day); this was equivalent to 1.068 g of the crude drug.
Content determination and dissolution testing showed that the content of amygdalin in each dispersible tablet was 0.257 mg. In the dissolution test, the dissolution rates of the six batches of dispersible tablets at 50 min were 98%, 99%, 96%, 97%, 97%, and 98%, respectively, thus indicating that the dissolution rate of dispersible Taohong Siwu tablets was good.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64809/64809fig01.jpg" /> 
Figure 1: The mean trend for the test indices. The average trend chart for the test indicators. Factor A in the figure is the content of Taohong Siwu medicine powder, factor B is the content of PVPP, factor C is the content of CMS-Na, and factor D is the blank error group. The numbers in each factor represent different contents (see Table 3 for details). The value on the y-axis represents the K value, and lower K values indicate better results. <a href="https://www.jove.com/files/ftp_upload/64809/64809fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64809/64809fig02.jpg" /> 
Figure 2: Laetrile standard curve. The standard curve of amygdalin, with the x-axis representing the injection volume, and the y-axis representing the peak area. <a href="https://www.jove.com/files/ftp_upload/64809/64809fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64809/64809fig03.jpg" /> 
Figure 3: Drug load screening results. This figure shows the drug load results; the left y-axis is the time, the right y-axis is the hardness, and the four prescriptions on the x-axis represent different drug load volumes. <a href="https://www.jove.com/files/ftp_upload/64809/64809fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>serial number</td>
			<td>medicinal powder (g)</td>
			<td>PVPP (g)</td>
			<td>MCC (g)</td>
			<td>pregelatinized starch (g)</td>
			<td>lactose (g)</td>
			<td>Disintegration time limit (s)</td>
			<td>Exterior</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.5</td>
			<td>1</td>
			<td>3.4</td>
			<td></td>
			<td></td>
			<td>26</td>
			<td>clean</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.5</td>
			<td>1</td>
			<td></td>
			<td>3.4</td>
			<td></td>
			<td>54</td>
			<td>clean</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.5</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td>3.4</td>
			<td>16</td>
			<td>common</td>
		</tr>
	</tbody>
</table>
Table 1: Filler selection results. The main drug content and PVPP dosage in the three prescriptions remained unchanged; prescription 1 used MCC as the filler, prescription 2 used pregelatinized starch as the filler, and prescription 3 used lactose as the filler. From these, lactose as the filler had the shortest disintegration time, but the gloss was not up to standard.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Disintegrant prescription</td>
			<td>Exterior</td>
			<td>Disintegration time limit (s)</td>
			<td>Dispersion uniformity (s)</td>
		</tr>
		<tr>
			<td>PVPP+L-HPC</td>
			<td>clean</td>
			<td>39</td>
			<td>39</td>
		</tr>
		<tr>
			<td>L-HPC+CMS-Na</td>
			<td>clean</td>
			<td>40</td>
			<td>52</td>
		</tr>
		<tr>
			<td>PVPP+CMS-Na</td>
			<td>clean</td>
			<td>42</td>
			<td>40</td>
		</tr>
	</tbody>
</table>
Table 2: Disintegrant combination screening. Under conditions with the main drug and filler remaining unchanged, different disintegrant combinations were tested in terms of their disintegration time limit and dispersion uniformity. Prescription 1 was PVPP + L-HPC, prescription 2 was L-HPC + CMS-Na, and prescription 3 was PVPP + CMS-Na, among which the combination of PVPP + CMS-Na had the shortest disintegration time limit.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Level</td>
			<td>A&#160; (g)</td>
			<td>B (g)</td>
			<td>C (g)</td>
			<td>D (g)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>3.4</td>
			<td>0.6</td>
			<td>0.4</td>
			<td>blank</td>
		</tr>
		<tr>
			<td>2</td>
			<td>4.4</td>
			<td>1.2</td>
			<td>0.8</td>
			<td>blank</td>
		</tr>
		<tr>
			<td>3</td>
			<td>5.4</td>
			<td>1.8</td>
			<td>1.2</td>
			<td>blank</td>
		</tr>
	</tbody>
</table>
Table 3: Factor levels for orthogonal designs. The dosage of the main drug of each prescription remained unchanged. Factor A is the dosage of filler MCC, factor B is the dosage of PVPP, factor C is the dosage of CMS-Na, and factor D is the blank error.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Numbering</td>
			<td>A (g)</td>
			<td>B (g)</td>
			<td>C (g)</td>
			<td>D (blank)</td>
			<td>Disintegration time limit (s)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>69</td>
		</tr>
		<tr>
			<td>2</td>
			<td>1</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
			<td>123</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1</td>
			<td>3</td>
			<td>3</td>
			<td>3</td>
			<td>40</td>
		</tr>
		<tr>
			<td>4</td>
			<td>2</td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>43</td>
		</tr>
		<tr>
			<td>5</td>
			<td>2</td>
			<td>2</td>
			<td>3</td>
			<td>1</td>
			<td>31</td>
		</tr>
		<tr>
			<td>6</td>
			<td>2</td>
			<td>3</td>
			<td>1</td>
			<td>2</td>
			<td>39</td>
		</tr>
		<tr>
			<td>7</td>
			<td>3</td>
			<td>1</td>
			<td>3</td>
			<td>2</td>
			<td>78</td>
		</tr>
		<tr>
			<td>8</td>
			<td>3</td>
			<td>2</td>
			<td>1</td>
			<td>3</td>
			<td>59</td>
		</tr>
		<tr>
			<td>9</td>
			<td>3</td>
			<td>3</td>
			<td>2</td>
			<td>1</td>
			<td>34</td>
		</tr>
		<tr>
			<td>K1</td>
			<td>232</td>
			<td>190</td>
			<td>167</td>
			<td>134</td>
			<td></td>
		</tr>
		<tr>
			<td>K2</td>
			<td>113</td>
			<td>213</td>
			<td>200</td>
			<td>240</td>
			<td></td>
		</tr>
		<tr>
			<td>K3</td>
			<td>171</td>
			<td>113</td>
			<td>149</td>
			<td>142</td>
			<td></td>
		</tr>
		<tr>
			<td>K1</td>
			<td>77.333</td>
			<td>66.333</td>
			<td>55.667</td>
			<td>44.667</td>
			<td></td>
		</tr>
		<tr>
			<td>K2</td>
			<td>37.667</td>
			<td>71</td>
			<td>66.667</td>
			<td>80</td>
			<td></td>
		</tr>
		<tr>
			<td>K3</td>
			<td>57</td>
			<td>37.667</td>
			<td>49.667</td>
			<td>47.333</td>
			<td></td>
		</tr>
		<tr>
			<td>R</td>
			<td>39.667</td>
			<td>33.333</td>
			<td>17</td>
			<td>35.333</td>
			<td></td>
		</tr>
		<tr>
			<td>primary and secondary</td>
			<td>RA&#62;RD&#62;RB&#62;RC</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 4: Orthogonal experimental arrangement and experimental results.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>source of variance</td>
			<td>sum of squared deviations</td>
			<td>degrees of freedom</td>
			<td>mean square</td>
			<td>F value</td>
			<td>salience</td>
		</tr>
		<tr>
			<td>A</td>
			<td>236.667&#160;</td>
			<td>2</td>
			<td>1180.333</td>
			<td>1.016</td>
			<td>&#62;0.05</td>
		</tr>
		<tr>
			<td>B</td>
			<td>1828.667</td>
			<td>2</td>
			<td>914.333</td>
			<td>0.787</td>
			<td>&#62;0.05</td>
		</tr>
		<tr>
			<td>C</td>
			<td>446</td>
			<td>2</td>
			<td>223</td>
			<td>0.192</td>
			<td>&#62;0.05</td>
		</tr>
		<tr>
			<td>D (error)</td>
			<td>2322.667</td>
			<td>2</td>
			<td>1161.333</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 5: Variance analysis results. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Prescription</td>
			<td>Medicinal powder (g)</td>
			<td>MCC (g)</td>
			<td>PVPP (g)</td>
			<td>CMS-Na (g)</td>
			<td>Magnesium stearate (g)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>1</td>
			<td>4.4</td>
			<td>1.8</td>
			<td>1.2</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>2</td>
			<td>1.5</td>
			<td>4.4</td>
			<td>1.8</td>
			<td>1.2</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>3</td>
			<td>2</td>
			<td>4.4</td>
			<td>1.8</td>
			<td>1.2</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>4</td>
			<td>2.5</td>
			<td>4.4</td>
			<td>1.8</td>
			<td>1.2</td>
			<td>0.1</td>
		</tr>
	</tbody>
</table>
Table 6: Drug load formulations. The dosage of MCC, PVPP, and CMS-Na for each prescription remained unchanged. The dosage of the main drug in prescription 1 was 1 g, in prescription 2 was 1.5 g, in prescription 3 was 2 g, and in prescription 4 was 2.5 g.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Batch number</td>
			<td>Exterior</td>
			<td>Average sheet weight (g)</td>
			<td>Weight difference (g)</td>
			<td>Average hardness (N)</td>
			<td>Disintegration time limit (s)</td>
			<td>Dispersion uniformity (s)</td>
		</tr>
		<tr>
			<td>20220710</td>
			<td>clean</td>
			<td>0.1978</td>
			<td>qualified</td>
			<td>22</td>
			<td>39</td>
			<td>43</td>
		</tr>
		<tr>
			<td>20220711</td>
			<td>clean</td>
			<td>0.186</td>
			<td>qualified</td>
			<td>21</td>
			<td>35</td>
			<td>41</td>
		</tr>
		<tr>
			<td>20220712</td>
			<td>&#160;clean&#160;</td>
			<td>0.1948</td>
			<td>qualified</td>
			<td>18</td>
			<td>29</td>
			<td>32</td>
		</tr>
	</tbody>
</table>
Table 7: Quality evaluation for the dispersible Taohong Siwu tablets. An appearance analysis, average weight analysis, weight difference check, hardness check, disintegration time limit check, and dispersion uniformity check were performed for the three batches of samples.

Watch this Scientific Journal Video about Formation of Dispersible Taohong Siwu Tablets at JoVE.com

Formation of Dispersible Taohong Siwu Tablets

This article discusses the process used to formulate and prepare Taohong Siwu, a traditional Chinese medicine, as a dispersible tablet. We performed single-factor and orthogonal experiments to screen the prescription excipients, optimize the formulation process, and produce dispersible Taohong Siwu tablets.

Here, we optimize the process used to formulate and prepare dispersible Taohong Siwu tablets and provide a foundation for expanding their clinical ...

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1.4K Views.  The First Affiliated Hospital of Anhui University of Chinese Medicine. This article discusses the process used to formulate and prepare Taohong Siwu, a traditional Chinese medicine, as a dispersible tablet. We performed single-factor and orthogonal experiments to screen the prescription excipients, optimize the formulation process, and produce dispersible Taohong Siwu tablets.

Video: Formation of Dispersible Taohong Siwu Tablets

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

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 ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

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.