The authors acknowledge funding from the NIDCR (Grant No. DE019902) and from the Israeli Science Foundation (Grant No. 382/13).

The protocol follows the guidelines of the institutional animal care and use committee (IACUC) of The Hebrew University of Jerusalem, Israel (Request No. MD-12-13524-4), an AAALAC approved facility, and by the Cedars-Sinai Medical Center IACUC (Request No. 3770). The animals were treated in strict adherence to NIH guidelines.
1. Preparation of Bone Allografts
<ol>
	<li>Euthanize 7- to 8-week-old Balb/C mice, or any strain different from the recipient, using the standard method of CO2 inhalation or intraperitoneal injection of 50 &#956;l phenobarbital (65 mg/ml.; 100 mg/kg). Confirm the absence of a heartbeat and perform cervical dislocation. Alternatively, harvest the allografts from carcasses that have been kept in &#8211;20 &#176;C and thawed prior to harvest.</li>
	<li>Shave the calvarial region using a hair clipper, applying it against the direction of hair growth. Disinfect the carcass head by applying 70% isopropanol. Cut the scalp skin using a No. 11 scalpel and remove the periosteum.</li>
	<li>Using a dental drill and a 4.5-mm inner-diameter trephine, detach a circular fragment of calvarial bone. Transfer the bone fragment to a 100-mm plastic dish containing sterile saline. At this point, observe blood and soft tissue attached to the bone fragment (Figure 1A). Obtain bone fragments in this manner from 22 donors.</li>
	<li>Hold one bone fragment at a time using curved tweezers. Swab it thoroughly using cotton tipped applicator and remove any brain tissue, soft tissue, and blood (Figure 1B). Gently cut away any bone chips that remain attached to the graft using fine scissors so that the graft will have a perfect rounded shape.</li>
	<li>Dip each graft once in a 15 ml tube containing 70% ethanol and twice in 15 ml tubes containing phosphate-buffered saline to wash the ethanol.</li>
	<li>Freeze the allografts in a &#8211;80 &#176;C refrigerator in cryotubes for at least 1 week. At this point, make sure the allografts appear transparent (Figure 1C).</li>
</ol>
2. Calvarial Defect Surgery
<ol>
	<li>For recipients use transgenic mice that express luciferase under the control of the osteocalcin promoter6.</li>
	<li>Sterilize the tips of the surgical tools using a glass bead heater for 30 sec. Transfer the tools to a jar containing 70% isopropanol to maintain sterile conditions. Use sterile gloves.</li>
	<li>Anesthetize a 7- to 8-week-old female mouse by intraperitoneal injection of a ketamine- medetomidine mixture (75 mg/kg and 1 mg/kg, respectively). Pinch the animal limb to make sure it is deeply anesthetized. Do not leave an animal unattended until it regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Shave the calvarial region using a hair clipper by applying it against the direction of hair growth. Apply hair-removing cream to remove any fur remnants and wipe it after no longer than 2 min using dry gauze followed by saline soaked gauze. Care should be taken to avoid getting it in the eyes. Alternatively, the vet eye ointment can be applied prior to hair removal as an extra layer of protection for the eyes.&#160;It is imperative that all traces of the depilatory cream be removed in order to avoid possible irritation from excessive exposure to the chemical agent. Disinfect the scalp by applying 5% chlorhexidine (Figure 1D).</li>
	<li>Inject subcutaneously 5 mg/kg carprofen diluted in 200 &#181;l saline. Apply vet eye ointment to prevent dryness and keep the animal on a thermal pad at all times. Position the animal under the operating binoculars.</li>
	<li>Make a 1-cm-long oval cut in the scalp skin using fine scissors and tweezers (Figure 1E). Hold the periosteum using curved tweezers and cut away using fine scissors.</li>
	<li>Drill the calvarial defect 2 mm anterior to the lambdoid suture by using a dental drill and a 5-mm outer-diameter trephine (Figure 1F and G). To avoid penetration of the dura mater, drill three-fourths of the way into the bone and detach the bone fragment using a spatula.</li>
	<li>To implant an autograft, retrieve the bone fragment and wash it in a 10 mm plastic plate containing 10 ml sterile saline to remove debris. Do not remove the soft tissues. To implant an allograft, thaw in advance an allograft&#160;and normalize it to RT, and then wash it.</li>
	<li>Place the bone graft in the defect using curved tweezers so it will not touch the defect margins ( Figure 1H). Glue the graft to the host bone using fibrin gel (5 &#181;l of 46 mg/ml fibrinogen mixed with 5 &#181;l of 25 U/ml thrombin).</li>
	<li>Suture the skin using 4-0 vicryl suture ( Figure 1I). Apply povidone-iodine to the surgical cut. Care should be taken to prevent the contamination of the suture by fur.&#160;Mark the mouse ear and inject 0.25 mg/kg Antisedan to reverse the anesthetic effect of medetomidine.</li>
	<li>If the animal does wake up in 10 min, make sure it is situated on a warmed pad. Inject 200 &#181;l sterile saline, and if needed inject 0.1 mg/kg Antisedan. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Keep the animals in separated cages for a week to prevent them from opening the sutures. Place mouse chow soaked in water in a petri dish on the cage floor for a few days post-op.&#160;Inject subcutaneously 200 &#181;l of 1mg/ml Carprofen solution diluted in sterile saline once a day for 3 days after surgery to treat post-surgical pain.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53459/53459fig1.jpg" /> 
Figure 1.&#160;Calvarial Allograft Preparation and Calvarial Defect Surgery. The bone fragment is isolated from the calvarial region (A). Soft tissue, bone marrow, and blood are swabbed (B), and the graft is rinsed in 70% ethanol followed by two washes in phosphate-buffered saline (C, AG = allograft). The recipient mouse is prepared for surgery by shaving and disinfection of skin in the calvarial region (D). An oval cut is created in the scalp skin, and the periosteum is removed (E). Next, the calvarial defect is drilled using a 5-mm-diameter trephine (F). The bone fragment is detached using a spoon-shaped spatula, leaving the superior sagittal sinus intact (G). The allograft is then placed in the defect and secured using fibrin gel (H). Finally, the skin is sutured (I). <a href="https://www.jove.com/files/ftp_upload/53459/53459fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Micro-Computed Tomography for Characterization of the Vascular Tree
<ol>
	<li>Prepare heparinized saline. Weight 2,000 U sodium heparin and place it in 50ml plastic tube. Add 20 ml saline and shake well the tube. Fill a 20-ml Luer lock syringe with 15 ml warmed heparinized saline and connect the syringe to a 23-G scalp vein set. Affix the syringe to a syringe pump, and set it to pump 10 ml at 1 ml/min.</li>
	<li>Sedate a mouse using an intraperitoneal injection of 50&#181;l phenobarbital (65 mg/ml.; 100 mg/kg). Pinch the animal&#8217;s limb to make sure it is deeply sedated. Secure the mouse on its back to a fixation board wrapped with an absorbent surface liner (Figure 2A).&#160;Place the fixed mouse in a fume hood to prevent personnel exposure to fume.</li>
	<li>Cut the skin from the abdomen to the neck using fine scissors and tweezers (Figure 2B). Cut the abdominal wall up to the xiphoid process (Figure 2C). Cut the rib cage along its lateral aspect. Avoid injuring the lungs and heart, and make sure the heart is fully exposed. Use a hemostat to retract the sternum (Figure 2D).</li>
	<li>Insert the butterfly needle 3 mm into the left ventricle (Figure 2E). Glue the needle to the heart and chest wall using 3-sec glue (Figure 2F).</li>
	<li>Activate the pump immediately. After a minute, dissect the inferior vena cava in order to depressurize the vasculature (Figure 2G). Tilt the board so that liquids will flow away from the head. Successful perfusion will result in clearing the liver and blood vessels.</li>
	<li>When the heparinized saline perfusion is completed, perfuse 10 ml&#160;of 4% formaldehyde. Avoid penetration of air bubbles into the vasculature and avoid exposure to formaldehyde fumes. Successful formaldehyde perfusion will result in stiffening of the tail. Flush out the formaldehyde with 10 ml heparinized saline.</li>
	<li>Prepare the radio-opaque contrast agent according to manufacturer instructions. Typically, it will require mixing of the compound, diluent, and a curing agent. Use serological pipettes and mix the solution in a 15 ml plastic tube.</li>
	<li>Perfuse the animal with the contrast agent mixture at a rate of 1 ml/min. Observe the coronary,&#160;intestinal and hepatic blood vessels and make sure they are turning yellow after perfusion of 2 - 3 ml, indicating successful perfusion (Figure 2H). Upon completion of perfusion, cut the butterfly needle&#8217;s tubing, obtain the carcass alone and wrap it with toilet paper to prevent leakage of the solution to the calvarial region. Allow polymerization in 4 &#176;C O/N.</li>
	<li>Dissect the calvarial region (Figure 2I); First use a scalpel to cut the skin as lateral as possible, avoiding damaging the cranial region of interest. Locate the bone graft and then use fine scissors to cut the cranial bone 10 mm away from the graft.</li>
	<li>Scan the isolated calvarial bone sample using a &#956;CT scanner; set field of view to 20.5 mm, X-ray energy of 55 kVp, Intensity of 145 &#956;A, using 1,000 projections per 360&#176; and integration time of 200 msec. Adjust &#956;CT parameters7 to image other bone defect models.</li>
	<li>Observe the 2D reconstructed slices that were generated by the &#956;CT software. Make sure to scan the region of interest is properly and then locate the calvarial defect. Assess the quality of the perfusion. After successful identification of blood vessels&#160;(Figure 2J), continue and decalcify the sample using 6% Trichloroacetic Acid (TCA) diluted in Double Distilled Water (DDW).</li>
	<li>Rescan the sample using the parameters indicated in Section 3.10. Locate the Lambdoid suture in the reconstructed 2D slices. Define a cylindrical volume of interest (VOI) in height of 2 mm and 8 mm diameter, that is tangent to the suture &#8211; this is the anatomical site of defect.</li>
	<li>Segment the bone tissue from soft tissues using a global thresholding procedure; set the lower threshold to 130, and apply a constrained 3D Gaussian filter (sigma [&#963;] = 2.0 and support = 2) to partially suppress noise in the volumes. Generate a 3D reconstruction and make sure the VOI is properly positioned (Figure 2K).</li>
	<li>Generate a thickness map using the IPL software and the &#8220;dt_object_param&#8221; function (Figure 2L). 
	NOTE:This function describes the vessel volume for each vessel diameter.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53459/53459fig2.jpg" /> 
Figure 2.&#160;Perfusion using a radio-opaque agent via the cardiac approach. The mouse is affixed to the fixation board (A), and the skin is cut along the abdomen up to the neck (B). The abdominal wall is cut along the medial line up to the xiphoid process (C), and the rib cage is cut laterally (D). A butterfly needle is inserted into the left ventricle, avoiding insertion into the right ventricle, which is markedly darker (E, the right ventricle is indicated by the white dashed line), and the needle is affixed to the heart and chest wall (F). Heparinized saline (2&#8211;3 ml) is pumped into the heart, and the inferior vena cava is dissected (G, white arrow indicates the inferior vena cava). Formaldehyde (4%) is perfused and washed out; this is followed by a perfusion of yellow-dyed radio-opaque contrast agent. Successful perfusion will be evident by clearance of the blood vessels, which change color to yellow (H). After polymerization, the calvarial region is isolated (I, AG = allograft) and imaged using a &#181;CT scanner to confirm proper perfusion (J, volume of interest is marked with orange). The sample is decalcified and rescanned (K), followed by generation of a colored thickness map (L). <a href="https://www.jove.com/files/ftp_upload/53459/53459fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Fluorescence Imaging of Bone Mineralization and Blood Perfusion
<ol>
	<li>Reconstitute probes to a concentration of 2 nmol/100 &#181;l PBS in a 1.5 ml tube (use a bisphosphonate-conjugated fluorescent probe to monitor bone mineralization and a blood-borne fluorescent agent to measure blood perfusion). Pipette gently several times to ensure homogenous dissolving of the probe.</li>
	<li>24 hr before the designated imaging session take a time-zero image; anesthetize 3 mice using 2 L/min inhalation of 100% medical-grade oxygen supplemented with 3% isoflurane in an induction chamber. After proper anesthesia has been established, lower the isoflurane concentration to 1.5% &#8211; 2%.</li>
	<li>Apply vet eye ointment to prevent dryness and make sure thermal pad is switched on at all times. Pinch the animal limb to make sure it is deeply anesthetized. Do not leave an animal unattended until it regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Shave the calvarial region using a hair clipper applying it against the direction of hair growth. Remove fur remnants using hair-removing cream. Any fur remnant will interfere with optical imaging. Remove sutures using fine scissors and tweezers.</li>
	<li>Place three animals on the IVIS stage warmed to 37 &#176;C for each imaging session. Set exposure time to auto, view field to C, and adjust filters. e.g., for a bisphosphonate-conjugated fluorescent probe characterized with maximal excitation at light wavelength of 680 nm, use an excitation filter of 675 nm and an emission filter of Cy5.5. Similarly, when using a blood-borne fluorescent agent with peak excitation at light wavelength 750 nm use an excitation filter of 710 nm and an emission filter of 780 nm.</li>
	<li>Image the mice through Living Image software by pressing the &#8220;Acquire&#8221; button. Make sure the calvarial region is completely visible (Figure 3B) For a comprehensive guide advise Lim et al 20098.</li>
	<li>Allow the mice to awake inhaling 100% oxygen.</li>
	<li>Inject the 2-nmol fluorescent probe via an intravenous tail injection. At the designated imaging session, perform imaging as described above.</li>
</ol>
5. Micro-Computed Tomography for Bone Regeneration
<ol>
	<li>For quantification of bone formation, anesthetize the mouse by inhalation of 3% isoflurane, as depicted in step 4.2 - 4.3. Transfer the mouse to the &#956;CT scanner bed, and place it in the scanner.</li>
	<li>Set the &#956;CT scanner&#8217;s x-ray tube potential energy to 55 kVp and intensity of 145 &#181;A medium resolution. Using a field of view of 20.5 mm, perform a scout (single x-ray) scan and define a region of interest extending from the mouse&#8217;s eyes to the back of its skull. Perform a CT scan, using 1,000 projections per 360&#176; and integration time of 200 msec.</li>
	<li>Reconstruct the 2D slices using a nominal spatial resolution of 20 &#181;m. Align the defect margins to a standardized position using &#956;CT software9.</li>
	<li>Similar to step 3.20, define a cylindrical volume of interest and apply the constrained 3D Gaussian filter (&#963; = 0.8 and support = 1) to partially suppress noise in the volumes. Segment the bone tissue from soft tissues using a global thresholding procedure.</li>
	<li>Determine the volume of mineralized bone tissue in the volume of interest.</li>
</ol>
6. Bioluminescence Imaging of Stem Cell Recruitment and Differentiation
<ol>
	<li>Prepare the mouse for imaging as depicted in Sections 4.2-4.4 and awake the mice by inhalation of 100% oxygen.</li>
	<li>Administer 126 mg/kg beetle luciferin to each mouse via intraperitoneal injection, and return the animal to the induction chamber. After 2 min, anesthetize the animal using 3% isoflurane. Place the mice on the IVIS warmed stage.</li>
	<li>Via Living Image software, set the exposure time to &#8220;auto,&#8221; and the field of view to C. Image the mice 5 min after luciferin administration. Image the animals as depicted in step 4.6, using the &#8220;Bioluminescence&#8221; modality Define regions of interest because transgene expression varies between mice. Normalize the calvarial signal to the tail.</li>
</ol>

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Endocrinology. 143 (4), 1545-1553 (2002).</li><li>Cornejo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+adipose+tissue-derived+osteogenic+and+endothelial+cells+on+bone+allograft+osteogenesis+and+vascularization+in+critical-sized+calvarial+defects.">Effect of adipose tissue-derived osteogenic and endothelial cells on bone allograft osteogenesis and vascularization in critical-sized calvarial defects.</a> Tissue Eng Part A. 18 (15-16), 1552-1561 (2012).</li></ol>

The authors have nothing to disclose.

The aim of the multimodal imaging approaches described here is to enable meticulous investigation of the angiogenesis-osteogenesis axis in the context of cranial bone grafting. Neovascularization was imaged using a &#181;CT protocol, which allowed an accurate high-resolution 3D demonstration of the vascular tree feeding the entire cranial defect. &#181;CT data can be readily analyzed using advanced tools such as IPL software. For example, the thickness analysis shown in Figure 3C revealed that the main d...

Craniofacial bone loss due to trauma, tumor resection, decompressive craniotomy, and congenital defect rarely heals by itself and presents a clear unmet clinical need. Autologous bone grafts and allogeneic bone grafts are extensively used to treat these conditions1.
It is widely accepted that osteogenesis is tightly coupled with angiogenesis2,3. Thus, the complete study of a proposed therapy for bone regeneration should include a comprehensive investigation of the vascular tree forming throughout the entire defect site. There are several available methods to characterize vascularization in research models. The vascular tree can be investigated by histological analysis. Since histology relies on sectioning tissue, there is a high probability that the resulting image will be distorted. To address this problem, intravital microscopy can be performed to image intact blood vessels4; however, this method is limited to one-plane imaging. &#181;CT scanning of specimens obtained from an animal perfused with contrast agent allows 3D imaging of the vascular network that feeds the regeneration site5. This approach allows a highly detailed demonstration of an organ&#8217;s vasculature as a whole, as well as a meticulous analysis of blood vessel distribution. Furthermore, &#181;CT enables differentiation between varied diameters of blood vessels, which characterize the different subtypes of blood vessels.
We hypothesized that implantation of a calvarial autograft will induce greater neovascularization than implantation of an allograft, and this increased neovascularization will lead, in turn, to enhanced bone formation.To pursue this hypothesis we employed a variety of techniques. We investigated patterns of the newly formed vascular tree by performing a &#181;CT-based analysis. We measured blood perfusion using a blood-pool fluorescent probe. Next, we assessed bone tissue mineralization by FLI of a hydroxyapatite-directed probe and &#181;CT analysis. Finally, we monitored stem cell recruitment and differentiation, performing BLI in transgenic mice in which luciferase is expressed in osteocalcin-positive cells.

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			<td height="42" style="height:42px;width:216px;">C57BL/C&#160; Mice</td>
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			<td height="42" style="height:42px;width:216px;">FVB/n Mice</td>
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			<td align="right" style="width:119px;">862</td>
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			<td height="42" style="height:42px;width:216px;">Phenobarbital</td>
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			<td style="width:119px;">NDC 0641-0477-25</td>
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			<td height="21" style="height:21px;width:216px;">Rodent hair clipper</td>
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			<td height="21" style="height:21px;width:216px;">Scalpel 11</td>
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			<td height="21" style="height:21px;width:216px;">Domitor</td>
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			<td style="width:119px;">NADA 141-267</td>
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			<td height="21" style="height:21px;width:216px;">carprofen</td>
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			<td style="width:119px;">02000/4229</td>
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			<td height="21" style="height:21px;width:216px;">Operating binocular</td>
			<td style="width:112px;">Kent scientific</td>
			<td style="width:119px;">KSCXTS-1121</td>
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			<td height="42" style="height:42px;width:216px;">Fine scissors&#160;</td>
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			<td style="width:119px;">14060-11</td>
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			<td height="42" style="height:42px;width:216px;">Curve tweezers</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spoon shaped spatula</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">10090-13</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Tisseel Fibin gel kit&#160;</td>
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			<td height="42" style="height:42px;width:216px;">needle holder</td>
			<td style="width:112px;">Fine Science tools</td>
			<td style="width:119px;">12060-01</td>
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			<td height="21" style="height:21px;width:216px;">vicryl suture 4-0</td>
			<td style="width:112px;">Ethicon</td>
			<td style="width:119px;">J392H</td>
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			<td height="21" style="height:21px;width:216px;">Antisedan</td>
			<td style="width:112px;">Zoetis</td>
			<td style="width:119px;">NADA#141033</td>
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			<td height="21" style="height:21px;width:216px;">Heparin</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">H3393</td>
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			<td height="21" style="height:21px;width:216px;">20ml luerlock&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Hemostat</td>
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			<td style="width:119px;">13008-12</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe pump</td>
			<td style="width:112px;">Harvard apparatus</td>
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			<td height="21" style="height:21px;width:216px;">3sec gel glue&#160;</td>
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			<td height="21" style="height:21px;width:216px;">rodent dissection board</td>
			<td style="width:112px;">Leica</td>
			<td style="width:119px;">38DI02313</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microfil MV-122</td>
			<td style="width:112px;">flow-tech</td>
			<td style="width:119px;">MV-122</td>
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			<td height="21" style="height:21px;width:216px;">uCT40 scanner</td>
			<td style="width:112px;">Scanco</td>
			<td style="width:119px;">uCT40</td>
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			<td height="21" style="height:21px;width:216px;">TCA6%</td>
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			<td style="width:119px;">T6399</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osteosense 680</td>
			<td style="width:112px;">PerkinElmar</td>
			<td style="width:119px;">NEV10020EX</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Angiosense750</td>
			<td style="width:112px;">PerkinElmar</td>
			<td style="width:119px;">NEV10011</td>
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			<td height="21" style="height:21px;width:216px;">Oxigen 100% medical grade</td>
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			<td style="width:119px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane (furane)</td>
			<td style="width:112px;">Baxter</td>
			<td align="right" style="width:119px;">1001936040</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS kinetics</td>
			<td style="width:112px;">Xenogen</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beetle luciferin</td>
			<td style="width:112px;">Promega</td>
			<td style="width:119px;">E160A</td>
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		</tr>
	</tbody>
</table>

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.

Neovascularization was assessed by &#181;CT volumetric analysis and by FLI using a fluorescent blood-borne agent to quantify blood perfusion. Seven days after surgery, &#181;CT scanning demonstrated a significantly higher volume of small- and medium-diameter blood vessels in mice that had received autografts than in mice that had received allografts harvested from C57BL/6&#160;(Figure 3A). Interestingly, in the autograft group the newly formed vascular tree appeared to reach the entire defect area, where...

Watch this Scientific Journal Video about Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts at JoVE.com

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

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 ...

computed-tomography-optical-imaging-osteogenesis-angiogenesis

Research

JoVE Journal

Bioengineering

11.4K Views.  The Hebrew University–Hadassah Faculty of Dental Medicine.  L'impianto di innesti ossei autologhi e allogenici costituiscono accettato approcci per trattare la perdita di massa ossea cranio-facciale. Eppure l'effetto della composizione trapianto sull'interazione tra neovascolarizzazione, la differenziazione cellulare e la formazione delle ossa non è chiaro. Vi presentiamo un protocollo di imaging multimodale mira a chiarire l'interdipendenza angiogenesi-osteogenesi in prossimità dell'innesto. 

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...