This work was carried out in part from support by the Hungarian Scientific Research Fund under Grant Number K112364, by the Hungarian Ministry of Human Capacities, Higher Education Excellence Program to Semmelweis University, Therapy Research Module and by the National Research, Development and Innovation Office KFI_16-1-2017-0409.&#160;The authors thank Dr. B&#225;lint Moln&#225;r, co-worker of the Department of Periodontology, Semmelweis University, who performed the vestibuloplasty in the presented clinical case.

The reported method was employed in a clinical trial which was granted ethical approval from the Hungarian Committee of the Health Registration and Training Center (approval number: 034310/2014/OTIG).
1. LSCI Setup
<ol>
	<li>Switch on the computer and any peripherals.</li>
	<li>Switch on the LSCI instrument to be used with the switch on the rear panel.</li>
	<li>Allow the instrument to warm up for at least 5 min. The instrument is ready for measurement when both LEDs on the rear panel have stopped flashing.</li>
	<li>Start the software by double-clicking on the software icon on the desktop or via the Start menu.</li>
	<li>Wait until both the yellow and the green LEDs on the rear panel have stopped flashing, which indicates that the laser is warm and initialization is finished. 
	Note: When starting up the system, one will occasionally be prompted to perform the verification procedure for the system.</li>
</ol>
2. System Verification
<ol>
	<li>Use the calibration box supplied. Remove the lid from the calibration box and shake it to avoid sedimentation in the colloidal suspension.</li>
	<li>Leave the lid off for 30 s to avoid bubbles.</li>
	<li>Put the lid back on the calibration box.</li>
	<li>Click Advanced | Verification | Verify instrument.</li>
	<li>Select Routine verification | Next.</li>
	<li>Turn the head 90&#176;, fasten the calibration box using the integrated magnets and click Next.</li>
	<li>Enter the room temperature in the text box, select&#160;&#176;C and click Start.</li>
	<li>Wait while the wizard completes the verification procedure.</li>
	<li>After a successful verification procedure close the wizard by clicking Finish.</li>
</ol>
3. Participant Preparation
<ol>
	<li>Make sure that measurement is performed in a temperature-controlled room (26&#160;&#176;C).</li>
	<li>Place the patient in a comfortable supine position in a dental chair and place a vacuum pillow under his head&#160;(Figure 1).</li>
	<li>Leave the patient undisturbed for 15 min before any measurements are taken.</li>
</ol>
4. Microcirculation Image Measurement
<ol>
	<li>In the Tools menu, select and click on Project Editor. A new window opens in which the commonly used settings can be saved.</li>
	<li>In the Projects box, click New to create a new project. Enter the &#8220;Vestibulum&#8221; and click OK.</li>
	<li>In the Sites box, click New to create a new site. Enter &#8220;Tooth 14&#8221; and click OK.</li>
	<li>Under the Contents of Tooth 14 panel add &#8220;10 cm&#8221; as the required distance for the Working Distance and enter a width of 3 cm and a height of 2 cm in the measurement boxes.</li>
	<li>Set the point density resolution to normal and the frame rate to 16 images/s and select Time from the Duration drop-down menu to set the recording duration to 0:30.</li>
	<li>Select &#8220;Record with no averaging&#8221; and set the color photo capture rate to 1/second.</li>
	<li>Then click &#8220;Apply&#8221; and &#8220;OK&#8221; to save the Project parameters.</li>
	<li>In the File menu, select and click&#160;New recording. A new Image Window will open&#160;and the Setup panel will be&#160;displayed.</li>
	<li>Under Recording Setup, select &#8220;Vestibulum&#8221; for the Project and &#8220;Tooth 14&#8221; for the 4.9. Site.</li>
	<li>Open the Subject drop-down menu, click New in the Select subject dialog box, and enter the name of the Patient.</li>
	<li>Click OK and enter a name for the recording in the Rec Name field: e.g., day 1 (days elapsed after the operation) and the name of the operator in the Operator field.</li>
	<li>Before beginning the microcirculation image measurement, measure patient&#8217;s blood pressure and pulse.</li>
	<li>Evacuate the air from the vacuum pillow to fix the patient&#8217;s head in a position appropriate to the area under investigation.</li>
	<li>Ask patient to open his mouth.</li>
	<li>Retract lips gently by two dental mirrors (Figure 1).</li>
	<li>Adjust the instrument&#8217;s head parallel to the measured area of the gingiva. A built-in visible (650 nm) indicator laser facilitates the positioning of the imager relative to the patient&#39;s mouth.</li>
	<li>Adjust the working distance to 10 cm by moving the instrument in relation to the tissue. The distance is measured continuously by the LSCI device and it is displayed by the software as working distance/measured value under Image Setup.</li>
	<li>Instruct the subject to remain still for the duration of the measurement.</li>
	<li>Click on the Record button to start recording. The color of the Image Window now changes to red, indicating that recording is in progress. The Setup panel is replaced by the Recording panel. Recording stops automatically after 30 s. When recording is finished, the color of the Image Window changes to blue and the Recording panel is replaced by the Review panel.</li>
	<li>Remove dental mirrors and allow the patient to close his mouth and swallow.</li>
	<li>Switch back to the live image by pressing the Resume recording button.</li>
	<li>Repeat the steps from 4.14&#160;to 4.21&#160;twice.</li>
	<li>Close the file. The data are saved automatically.</li>
	<li>Measure blood pressure and pulse after the LSCI measurements.&#160;</li>
</ol>
5. Offline Analysis
<ol>
	<li>Analyze the LSCI images using the built-in software. Go to Image or Split view&#160;(Figure 2).</li>
	<li>Define regions of interest (ROI). Note: the perfusion values of pixels within a ROI are averaged and defined as the blood flow value of the ROI, expressed in an arbitrary value called Laser Speckle Perfusion Unit (LSPU).</li>
	<li>Select the desired ROI shape within the ROI tools palette on the right.</li>
	<li>Select the Apply option in the ROI tools palette, which applies ROI operations to all images of the recording.</li>
	<li>Draw the ROI by clicking and holding the mouse button in the intensity image, dragging the ROI out to the desired size, and releasing the mouse button (click and double-click for free-form ROIs). Adjust the position of the ROI, resize or rotate it, if needed.</li>
	<li>Repeat steps from 5.3. to 5.5 as many times as the desired number of ROIs (Figure 3).</li>
	<li>Define time periods of interest (TOI). This allows for averaging perfusion in a ROI over a definite period of time (Figure 2).</li>
	<li>Go to Graph or Split view. Select the Add TOI tool button.</li>
	<li>Click and hold on the graph at the position where you want the TOI to begin and drag the cursor to the desired end position. Then release the mouse button.</li>
	<li>Export data from the mean value table for further processing.</li>
	<li>Construct blood flow curves by a suitable software used for statistical analysis.</li>
</ol>

The authors have nothing to disclose.

The aim of this study was to introduce a novel technique for monitoring the neovascularization of a graft in the human gingiva. According to our previous results, LSCI assesses the blood perfusion of the gingiva with good repeatability and reproducibility9, when strict implementation of each step of the planned protocol as a critical requirement is met. LSCI is regarded as a semi-quantitative technique that requires calibration periodically to ensure accuracy and stability. During verification, the room temperature must be measured as accurately as possible, because this value is used by the verification algorithm to calculate perfusion.
The LSCI method is highly sensitive to the working distance setting and movement artifacts as well. In this study, working distance was fixed at 10 cm. The measurement area was 2.7 cm x 2 cm, which corresponds to an approximately three teeth wide gingival area. The effective frame rate was 16 images/s and 0.06 s/image as the arterial pulse induces pulsatile changes in gingival microcirculation9, which has to be averaged out from the recording. Rapid imaging reduced the risk of movement artefacts, too. However, in case of incorrect settings or patient movements, the recording should be stopped and repeated under optimum conditions.
Two operators took part in every measurement: one adjusted the LSCI head and controlled the computer while the other retracted the lips of the patient. In this study, three repeated measurements were performed in each session, each taking 30 s. Since measurements always involve some kind of irritation to the soft tissue due to the inevitable retraction of the lips and cheeks, which disturbs the microcirculation of the gingiva, an increase in random error occurs. Such inter-day variation, however, can be minimized by repeating the entire measurement process, i.e., by re-opening the mouth, retracting the soft tissue again, re-setting the camera&#39;s position and re-selecting ROIs in the software9.
Gingival microcirculation showed high regional variation8. Therefore, a method such as LSCI which measures blood flow in a wide area has a great advantage over single point measurement techniques like LDF. In this study, the measurement area covered the whole surgical field. The wide measurement area allowed us to compare re- or neovascularization in various regions within the wounded area in our study. Contrary to LDF, where the probe is fixed by stents fabricated before surgery, in the case of the LSCI method, there is no need to define the region to be examined in advance. For the primary aim of monitoring wound healing in personalized medicine is to recognize unexpected patterns anywhere around the wound or the flap. In addition, postoperative changes in tissue geometry and edema caused by soft or hard tissue augmentation would make the pre-fabricated stents useless after surgery. To help visual evaluation, smoothing was turned on during recording and the smoothing value was set to 10. This means that perfusion was averaged over ten images for a smoother appearance of the perfusion image and in order to decrease background noise. However, smoothing is only a visual effect and does not influence actual recorded perfusion values.
Gingival blood flow has a high temporal variation as well. This may be related to many physiological factors which accompany everyday life, such as gingival inflammation16,17,18, circadian rhythm19, blood pressure20, temperature16,21, mechanical pressure8,22,23,24, tooth brushing14,17,25 or orthodontic force15. Therefore, the standardization and stabilization of these factors is obligatory for successful follow-up measurements.
The methods used earlier for investigating graft vascularization are highly invasive, which meant a major restriction on measurement time points during healing, especially in human studies26,27,28,29,30,31,32. They also have limitations in terms of measuring regional differences quantitatively. Our previous studies9,10 have already proved the high reliability of LSCI in clinical trials and it was found to be useful to determine the time of soft tissue healing of an individual after tooth extraction in order to optimize implant placement33. In this study, the wound area covered by a xenogenic collagen graft showed excellent neovascularization, as on the 11th postoperative day all zones within the graft achieved the maximum blood flow level. However, it could be presumed that the collagen graft sloughed off or was resorbed by day 11 and we actually measured the revascularization of the recipient bed. In addition to its non-invasive feature, another special attribute of LSCI is a capability to characterize reperfusion curves at various regions of a graft during incorporation at individual level. The centripetal characteristics of graft neovascularization are similar to previous histology observations30. This suggests that graft revascularization not only occurs from the periosteal vascular plexus but also from the wound margin.
The experiment presented shows that the revascularization of a graft can be clearly followed up if every step is followed strictly. However, on day 182, non-compliant patient preparation and instruction resulted in a significant increase in BF.
LSCI is extensively used for full-field imaging of vascular structure and associated blood flow in other tissues, like in the retina34,35, the skin7,36 and the brain37,38. The most promising clinical applications of LSCI are burn wound assessment39,40, evaluation of flaps41 and intraoperative cerebral blood flow monitoring42. Apparently, there are serious limitations to widespread gingival measurements by LSCI in human subjects. This tool is very robust and heavy. The main difficulties arise in connection with the documentation camera, which has low resolution and is located a few centimeters away from the measurement camera. These features make it difficult to identify regions of interest directly on color photos. The size of the LSCI machine head prevents shooting inside the oral cavity. Therefore, areas which are not visible directly may not be measured. We have demonstrated earlier that using an indirect approach with a photographic mirror may serve as an alternative method9. However, using a mirror involves more movement artifacts which LSCI is sensitive to, makes it more difficult to capture a perpendicular image and decreases the focal distance. When the measured area cannot be captured perpendicularly, the blood flow value may be correct9,39, but region identification on the image remains complicated because of 3D torsion.

Monitoring long-term changes of human gingival microcirculation in a clinical situation is a hot topic in oral and periodontal surgery. However, reliable assessment of perfusion can be difficult. There are only a few methods that do not invasively measure changes in the blood circulation of the human mucosa. Two of these employ a laser beam1,2,3,4, but in a different way. Laser doppler flowmetry (LDF) makes use of the Doppler shift in a laser beam5,6, while the laser speckle contrast imaging (LSCI) method relies on the speckle pattern of the backscattered laser light to measure the velocity of red blood cells7.
LDF measures only in a single point, and reproducible standardization of the sensors&#39; position is a desirable yet difficult task. Another problem is that the probe of the LDF is small in diameter (1 mm2). Measuring at predetermined points before surgery is too specific, and may be blind to postoperative circulatory changes, while edema, tissue removal, tissue movement or the implanted graft cause significant changes in the postoperative geometry of the affected soft tissue. The measuring distance of LDF is less than 1 mm which prohibits use of a dental splint with a predetermined hole for the probe in case of volumetric change of the tissue. LSCI does not require any special tools for localization and can measure in areas of several cm2. As a result, wound healing can be followed throughout the surgical site. In addition, LSCI can display blood perfusion in color-coded images at a fraction of a second, with a resolution of up to 20 &#956;m.
The LSCI device presented in this paper is used mostly for animal research applications where high resolution in small measurement areas is desired. However, since the structure and histology of the human oral mucosa are different from area to area (attached gingiva, marginal gingiva, vestibular mucosa), blood circulation is also heterogeneous8. Therefore, high-resolution LSCI has a great advantage over normal-resolution LSCI which is usually used in human testing.
The LSCI instrument employs an invisible laser (wavelength 785 nm). The beam is diverged to illuminate the measurement area, creating a speckle pattern. A CCD camera images the speckle pattern in the illuminated area. The CCD camera used in this system has an active imaging area of 1386 x 1034 pixels and its resolution is between 20&#8211;60 &#181;m/pixel depending on the size of the measurement area and on the setting of the software (low, medium, high). It can take images at a speed of 16 frames per second, or even more, up to 100 frames per second, if the image size is reduced. Blood perfusion is calculated by the built-in software. It analyzes variations in the speckle pattern and quantifies the contrast. The resulting flux is color coded to produce a perfusion image. According to our previous results, LSCI assesses the blood perfusion of the gingiva with good repeatability and reproducibility9. This implies that it is a reliable tool for monitoring changes in the microcirculation of the oral mucosa not only in short-term experiments, but also during long-term studies to track disease progression or wound healing10.
In this paper, we present a clinical case report to demonstrate that the high spatial resolution of LSCI makes it possible to reveal the neovascularization pattern of a xenogenic collagen graft. Furthermore, this case indicates that LSCI, owing to its high reliability, could sensitively detect individual variation. This is important as significant local anatomical variation and a different systemic background between the cases makes it difficult to standardize the surgical intervention in clinical trials of periodontal surgery.

<table><tbody><tr><td>PeriCam PSI-HR</td><td>Perimed AB, Stockholm, Sweden</td><td></td><td>The PeriCam PSI System is an imaging system based on LASCA technology (LAser Speckle Contrast Analysis). The system measures superficial blood perfusion over large areas at fast capture rates. This makes it ideal for investigations of both the spatial and temporal dynamics of microcirculation in almost any tissue.</td></tr><tr><td>PIMSoft</td><td>Perimed AB, Stockholm, Sweden</td><td></td><td>PIMSoft is a data acquisition and analysis software, intended for use together with the PeriCam PSI System and the PeriScan PIM 3 System, for measurement and imaging of superficial blood perfusion.</td></tr><tr><td>Geistlich Mucograft</td><td>Geistlich, Switzerland</td><td></td><td>It&#039;s a unique 3D collagne matrix designed specifically for soft tissue regeneration. It&#039;s indicated for the gain of keratinized tissue and recession coverage.</td></tr><tr><td>Omron M4</td><td>Omron Healthcare Inc., Kyoto, Japan</td><td></td><td>Blood pressure monitor, which gives accurate readings.</td></tr><tr><td>&lt;strong&gt;Nikon D5200&lt;/strong&gt;</td><td>Nikon Corportation, Tokyo, Japan</td><td></td><td>Taking intra oral photos</td></tr><tr><td>MS Excel</td><td>Microsoft Corporation, Redmond, Washington, USA</td><td></td><td>The software used for data management</td></tr><tr><td>IBM SPSS Statistics 25</td><td>IBM Corp., Armonk, NY, USA</td><td></td><td>The software used for statistical analysis</td></tr></tbody></table>

a novel approach to monitoring graft neovascularization in the human gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to increase vestibular depth, the zone of keratinized gingiva and soft tissue thickness for enhanced aesthetics and function. The apically repositioned split thickness flap combined with a collagen matrix is a frequently used vestibuloplasty procedure. Xenogenic collagen matrix is a viable alternative to autogenous gingival graft for increasing the amount of keratinized gingiva11,12,13; however, no data are available on the direction of graft revascularization and on how it affects the microcirculation of the surrounding tissues. Understanding these mechanisms may facilitate proper flap and incision design in periodontal surgery.
A 17 year-old male patient with an inadequate width of keratinized gingiva at the first premolar in the maxilla was treated by vestibuloplasty, using an apically repositioned split thickness flap combined with a collagen matrix. Intraoral photographs (taken by a photo camera) and blood flow (BF) measurements by LSCI were taken before the vestibuloplasty (baseline) as well as 1, 2, 3, 4, 5, 7, 9, 11, 14, 21, 27 days and 2, 3, 4, 5, 6 and 12 months postoperatively. Blood pressure and pulse were evaluated before and after each measurement.
During offline analysis, multiple ROIs were determined in the area of the augmented mucosa; some in the graft region and others in the surrounding mucosa, defined as &#39;peri&#39; regions. As shown in Figure 3, the &#39;peri&#39; and graft regions were further split into zones depending on distance from the center of the implanted graft, marked as zone F in the picture. Zones A and B were defined in the &#39;peri&#39; region and zones C, D and E in the graft region. Each of these zones was delimited separately at all four sides of the graft (mesial, distal, apical and coronal). Each 30-second shot was identified as a TOI (Figure 2). Data at each ROI and TOI were exported into a spreadsheet program. Blood flow curves were constructed by a suitable software used for statistical analysis.
There was no significant change in mean arterial pressure (MAP) during the one-year experiment, either in MAP before or after the blood flow measurements per session.&#160;Figure 4 shows a color photo, an intensity image and a perfusion image of the operated gingiva on the representative days of our study. During the first postoperative week complete flap closure, a thick layer of fibrin on the grafted area, and mild erythema and edema in the surrounding gingiva were visible. Blood perfusion images showed ischemia in the operated region and hyperemia in the &#39;peri&#39; regions. From day 14, the grafted area was clinically erythematous, in parallel with severe hyperemia observed on blood perfusion images. By the third month after graft incorporation, the wound healed, and gingival perfusion was close to preoperative circulation levels.
Figure 5 shows a blurred intensity image and the perfusion graph of the entire image. The sudden peak on the graph indicates movement by the patient. The measurement was repeated immediately, after making sure that the patient is in a comfortable position. Changes in BF in the different zones within the graft and in the &#39;peri&#39; regions are shown in Figure 6. It is common in all curves that from the fourth month, blood flow did not change any further until the end of the investigation. The average blood flow for this period could be used as a resting blood flow value for the new tissue and the random variation between time points allowed us to calculate the time-based variance component for each ROI respectively, using a linear mixed model. The minimum detectable difference could be calculated then to identify real change (with 95% confidence) between time points during the healing period (before the fourth month) in order to determine the hyperemic and the ischemic phase. The basic characteristics of curves were similar in all ROIs within the graft, starting with an ischemic phase followed by a hyperemic phase. However, the length of these two phases was different (Table 1). Ischemia was longest (7&#8211;9 days) in the central and in all coronal zones, with late hyperemia starting between day 11 and 27. In other zones of the graft, ischemia lasted only 4&#8211;7 days and hyperemia started earlier, between day 7 and 21.
The BF curves of the zones at the different sides of the graft had unique characteristics (Figure 6). At the apical side, all four zones had similar blood flow curves. At the coronal side, perfusion was regained in the outer zone later than in the inner zones, contrary to the mesial and distal side. At both lateral sides, BF increased first in zone C, then in zone D, followed by BF increase in zone E and finally in the central zone F. In the zones of the surrounding mucosa (zone A and B) no significant ischemia was observed. Instead, hyperemia of a different magnitude and extent was observed at the different sides.
There were two time points when the BF value did not tally with the overall characteristics of the BF curve. On day 9, there was a sudden drop in most zones and mainly in the &#39;peri&#39; zones of the apical and distal side. It cannot be stated with certainty that this was a measurement error, as no measurements were taken on the previous and on the following day. However, according to a note in the measurement report, the buccal fold was retracted with too much pressure by the operator, resulting in a drop in BF. This makes sense considering that mainly the circulation of the distal and apical sides could have been affected by pulling the cheek. On day 182 (6 months later), due to longer intervals between measurement times, the patient has forgotten to keep to the agreed restrictions before the measurement. The bleeding of the marginal gingiva on the colored photograph (Figure 6) indicates harsh tooth brushing prior to the measurement. In the meantime, the patient underwent orthodontic treatment, too, and he used intermaxillary elastics. Both factors could greatly increase BF14,15, so the measurement was repeated at a later time under more carefully controlled circumstances.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58535/58535fig1.jpg" /> 
Figure 1: Experimental LSCI setup and patient preparation for blood flow measurement in the operated area. The lips are retracted by dental mirrors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58535/58535fig1large.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/58535/58535fig2.jpg" /> 
Figure 2: Split view (combination of the Images view and the Graph view) of a typical recording of gingival blood flow in the treated area. Perfusion image (upper right sub-view) is a color-coded representation of blood perfusion in the gingiva. Areas of high perfusion are shown in red while areas of low perfusion are blue. The color range of perfusion images corresponds to 0-450 LSPU; smoothing was set to 10. An intensity image (lower right sub-view) is created by the total backscattered laser light. It corresponds exactly with the perfusion image and is useful for orientation and for identifying details in the perfusion image. Regions of interest (ROI) are always defined in the intensity image. The graph (upper left panel) shows real-time blood perfusion traces for each ROI in the recording. Check boxes to the left can be used to select which traces to show. Three consecutive measurements are shown on the graph. Each 30 s shot was identified as a TOI. A mean value table showing mean perfusion values in each ROI and TOI is also displayed in Split view (lower left panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58535/58535fig2large.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/58535/58535fig3.jpg" /> 
Figure 3: Regions of interest (ROI) defined within the examined gingival area in the intensity image. Zone A and B are in the &#39;peri&#39; region, while zone C, D and E are in the graft at decreasing distances from the center of the graft, marked as zone F. Zone A is situated on the vestibular surface of the lips. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58535/58535fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58535/58535fig4.jpg" /> 
Figure 4: Representative photographs (upper line), LSCI intensity image (middle line) and LSCI perfusion image (lower line) of the operated gingiva. The images represent the preoperative state and perfusion, and wound healing and perfusion 1, 4, 7, 14, 21, 27 and 98 days postoperatively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58535/58535fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58535/58535fig5.jpg" /> 
Figure 5: Split view of a sub-optimal recording. Blurred intensity image and outlying peaks on the graph as a result of incorrect setting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58535/58535fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58535/58535fig6.jpg" /> 
Figure 6: Scatter plot of BF over time at the coronal (a), mesial (b), distal (c) and apical (d) side of the graft. The central part of the graft (zone F) was depicted in all graphs to serve as a reference for more external zones. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58535/58535fig6large.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>Side</td>
			<td>Zone</td>
			<td>Ischemia end</td>
			<td>Hyperemia start</td>
			<td>Hyperemia end</td>
		</tr>
		<tr>
			<td>coronal</td>
			<td>c</td>
			<td>9</td>
			<td>27</td>
			<td>27</td>
		</tr>
		<tr>
			<td>coronal</td>
			<td>d</td>
			<td>9</td>
			<td>21</td>
			<td>27</td>
		</tr>
		<tr>
			<td>coronal</td>
			<td>e</td>
			<td>7</td>
			<td>11</td>
			<td>98</td>
		</tr>
		<tr>
			<td>central</td>
			<td>f</td>
			<td>9</td>
			<td>11</td>
			<td>98</td>
		</tr>
		<tr>
			<td>mesial</td>
			<td>c</td>
			<td>5</td>
			<td>21</td>
			<td>27</td>
		</tr>
		<tr>
			<td>mesial</td>
			<td>d</td>
			<td>5</td>
			<td>11</td>
			<td>61</td>
		</tr>
		<tr>
			<td>mesial</td>
			<td>e</td>
			<td>7</td>
			<td>11</td>
			<td>61</td>
		</tr>
		<tr>
			<td>distal</td>
			<td>c</td>
			<td>5</td>
			<td>11</td>
			<td>27</td>
		</tr>
		<tr>
			<td>distal</td>
			<td>d</td>
			<td>4</td>
			<td>7</td>
			<td>98</td>
		</tr>
		<tr>
			<td>distal</td>
			<td>e</td>
			<td>4</td>
			<td>11</td>
			<td>98</td>
		</tr>
		<tr>
			<td>apical</td>
			<td>c</td>
			<td>4</td>
			<td>11</td>
			<td>27</td>
		</tr>
		<tr>
			<td>apical</td>
			<td>d</td>
			<td>5</td>
			<td>11</td>
			<td>61</td>
		</tr>
		<tr>
			<td>apical</td>
			<td>e</td>
			<td>5</td>
			<td>11</td>
			<td>61</td>
		</tr>
	</tbody>
</table>
Table 1: Time frame of the ischemic and hyperemic phase in the various zones in the graft, in days

Watch this Scientific Journal Video about A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva at JoVE.com

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

6.7K Views.  Semmelweis University. Laser Speckle Contrast Imaging, of LSCI, kan helpen bij het monitoren van veranderingen op lange termijn in de menselijke gingivale microcirculatie in de kliniek om individuele ziekteprogressie en wondgenezing te volgen. De belangrijkste voordelen van deze techniek zijn dat het niet-invasief is en de bloedperfusie van het gingiva beoordeelt met een goede herhaalbaarheid en reproduceerbaarheid. Deze methode vereist geen speciaal gereedschap voor de lokalisatie van het meethoofd en de postopera...

Video: A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

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

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

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

Bioengineering

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

Developmental Biology

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

Biology

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

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