The authors would like to thank the Lam Lab at Georgia Tech for many helpful discussions in developing the experimental assays. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL115486 and by New Innovator Grant 1DP2OD007433-01 from the Office of the Director, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTE: The following protocol adheres to the guidelines set by the Institutional Review Board (IRB) at Georgia Tech.
1. Blood Collection and Red Blood Cell Isolation Procedure
<ol>
	<li>Collect 40-120 ml of blood from donors in 10 ml heparinized vacutainer tubes. Start PBMC (peripheral blood mononucleated cell) isolation within 4 hr of collection. During this time, keep blood at RT. 
	NOTE: O/N storage of blood in RT or in 4 &#176;C is NOT recommended as this will result in lower PBMC yield.</li>
	<li>Dilute whole blood 1:1 in ice-cold (4 &#176;C) sterile PBS.</li>
	<li>Pipette 10 ml of ice-cold hydrophilic polysaccharide into an empty, sterile 50 ml conical tube. Gently transfer diluted whole blood on top of the hydrophilic polysaccharide.</li>
	<li>Use a 25 ml pipette in slow setting and pipette blood along the side of the tube very slowly. Ensure that the blood is not mixed with the saccharide prior to centrifugation because the hydrophilic polysaccharide is toxic to cells and otherwise, the PBMC yield will be lower.</li>
	<li>Centrifuge blood samples for 30 min at 400 x g at 4 &#176;C. If available, reduce the brake speed of the centrifuge to half of maximum for better separation after centrifugation.</li>
	<li>Aspirate off the plasma/platelet fraction of the centrifuged blood sample. Carefully aspirate off the hydrophilic polysaccharide layer directly above the packed RBC layer.</li>
	<li>Collect 8-10 ml of packed RBCs and dilute 1:1 with unsterile 0.9% NaCl saline.</li>
</ol>
2. Confocal Microscopy Evaluation of Glycated Clot Structures (Simulated Diabetic Clots)
<ol>
	<li>Defrost human fibrinogen and fluorescently labeled human fibrinogen conjugate at 37 &#176;C.</li>
	<li>Pipette the following into a 0.5 ml graduated microcentrifuge tube.
	<ol>
		<li>For the healthy, normal fibrinogen clots, mix human fibrinogen with 10% fluorescently labeled human fibrinogen conjugate in a 50 mM Tris/100 mM NaCl buffer at a concentration of 5 mg/ml.</li>
		<li>For artificially glycated fibrinogen (to simulate diabetic clots), incubate human fibrinogen and 10% fluorescently labeled fibrinogen conjugate in a 5 mM solution of glucose dissolved in 50 mM Tris/100 mM NaCl for a resulting concentration of 6.8 mg/ml.</li>
	</ol>
	</li>
	<li>Cover the micro-centrifuge tubes using an opaque material to avoid exposure to light. Incubate the tubes in a 37 &#176;C water bath for 48 hr.</li>
	<li>Make a chamber on a glass microscope slide by affixing a thin adhesive on 2 sides of the slide.</li>
	<li>After the 48 hr incubation period, prepare reagents for fibrin formation by defrosting FXIIIa and human alpha thrombin at RT.</li>
	<li>Dilute FXIIIa to 80 U/ml in 5 mM CaCl2 buffer.</li>
	<li>Dilute thrombin to 4 U/ml in 5 mM CaCl2 buffer.
	<ol>
		<li>For normal clots, ensure that the final concentrations of fibrinogen, FXIIIa, and thrombin are 2.5 mg/ml, 20 U/ml, and 1 U/ml, respectively at a volume of 50 &#956;l.</li>
		<li>For the glycated clots, ensure that the final concentrations of fibrinogen, FXIIIa, and thrombin are 3.4 mg/ml, 20 U/ml, and 1 U/ml, respectively at a volume of 50 &#956;l.</li>
	</ol>
	</li>
	<li>Immediately pipette 30 &#956;l of the fibrin solution into the chamber formed by the adhesive.</li>
	<li>Place a 22 mm x 22 mm glass cover slip with thickness of 0.15 mm on top of the 2 layers of adhesive. Seal the open sides with clear adhesive to prevent the fibrin clot from drying out. Ensure that the adhesive does not interact with the fibrin clot.</li>
	<li>Allow the fibrin clots to polymerize at 21-23 &#176;C for 2 hr before confocal imaging.</li>
	<li>Take confocal microscopy images of the clots using a confocal microscope with a 40X/1.30 Oil M27 lens with a 488 nm Argon laser.</li>
</ol>
3. Confocal Microscopy Evaluation of Fibrin Clots Containing RBCs from Sickle Cell Patients
<ol>
	<li>Defrost human fibrinogen and fluorescently labeled human fibrinogen conjugate at 37 &#176;C.</li>
	<li>Pipette the following into a 0.5 ml graduated microcentrifuge tube.
	<ol>
		<li>For the healthy, normal fibrin clot, mix human fibrinogen with 10% fluorescently labeled human fibrinogen conjugate in a 50 mM Tris/100 mM NaCl buffer at a concentration of 5 mg/ml.</li>
		<li>For the clots containing SCD RBCs, mix human fibrinogen and 10% fluorescently labeled human fibrinogen conjugate in a 50 mM Tris/100 mM NaCl such that the resulting concentration of the fibrinogen is 10 mg/ml.</li>
	</ol>
	</li>
	<li>Label the isolated RBCs (both normal and those from sickle cell patients obtained from the RBC isolation protocol) using a cell-labeling solution.
	<ol>
		<li>Suspend cells at a density of 1 x 106 per ml in any chosen serum-free culture medium.</li>
		<li>Add 5 &#956;l/ml of the cell suspension to the cell-labeling solution. Mix well by gentle pipetting gently.</li>
		<li>Incubate for 20 min at 37 &#176;C.</li>
		<li>Centrifuge the labeled suspension tubes at 1,500 rpm for 5 min at 37 &#176;C.</li>
		<li>Remove the supernatant and gently re-suspend the cells in 37 &#176;C medium.</li>
		<li>Repeat the wash procedure (3.3.4 and 3.3.5) two more times.</li>
	</ol>
	</li>
	<li>Dilute FXIIIa to 80 U/ml in 5 mM CaCl2 buffer.</li>
	<li>Dilute thrombin to 4 U/ml in 5 mM CaCl2 buffer.
	<ol>
		<li>For normal clots, ensure that the final concentrations of fibrinogen, FXIIIa, and thrombin are 2.5 mg/ml, 20 U/ml, and 1 U/ml, respectively at a volume of 50 &#956;l.</li>
		<li>For the clots containing SCD RBCs, ensure that the final concentrations of fibrinogen, FXIIIa, and thrombin are 5 mg/ml, 20 U/ml, and 1 U/ml, respectively at a volume of 50 &#956;l.</li>
	</ol>
	</li>
	<li>Pipette 10 &#956;l of the labeled RBCs into the fibrin solution. Pipette 30 &#956;l of the fibrin with RBCs onto the microscope glass (refer to slide preparation for glycated clots).</li>
	<li>Allow the fibrin to polymerize at 21-23 &#176;C for 2 hr before confocal imaging.</li>
	<li>Take confocal images of the clots using the confocal microscope, and utilize excitation lasers with wavelengths of 488 nm and 633 nm to excite the fluorescently labeled fibrin and RBCs.</li>
</ol>
4. Confocal Microscopy Evaluation of Fibrinolysis Rates in Glycated Clot Structures
<ol>
	<li>Repeat the confocal microscopy protocol of glycated clots (Protocol Step 2) for the fibrinolysis rate evaluation in glycated clots.</li>
	<li>Do not seal the ends of the chamber with clear adhesive. Allow the fibrin clots to polymerize for 1.5 hr at 21-23 &#176;C in a sealed enclosure containing 250 ml of water to prevent dehydration.</li>
	<li>After polymerization, obtain images of the clots using the confocal microscope.</li>
	<li>Pipette plasmin into the open end of the chamber and disperse the plasmin through the clot via capillary action.
	<ol>
		<li>For the normal and glycated fibrinolysis experiments with normal concentrations, pipette 25 &#956;l at 200 &#956;g/ml of plasmin into the opening of the chamber.</li>
		<li>For the glycated clots with reduced concentrations, pipette 25 &#956;l at 50 &#956;g/ml into the opening of the chamber.</li>
	</ol>
	</li>
	<li>Use the time series feature (see Figure 1) in the confocal microscope software to capture real-time video footage of the plasmin lysing the clot.
	<ol>
		<li>Click acquisition mode and then select &#8220;Time Series.&#8221; Select the number of cycles and the recording time interval.</li>
	</ol>
	</li>
</ol>
5. Calculating Fibrinolysis Rates
<ol>
	<li>Determine the lysis rate by setting an area (2,500 &#181;m2) and calculating the elapsed time required to dissolve a fixed area of the clot. Calculate the lysis rate using the following equation (Figure 2): 
	<img alt="Equation 1" fo:content-width="2in" src="/files/ftp_upload/52019/52019eq1.jpg" /></li>
</ol>
6. Statistical Analysis of Fibrinolysis Rates
<ol>
	<li>Analyze the lysis data using statistical analysis software. Perform a one-way ANOVA and a post hoc Tukey-Kramer test44,45.</li>
</ol>

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+cell+disease+activates+peripheral+blood+mononuclear+cells+to+induce+cathepsins+k+and+v+activity+in+endothelial+cells.">Sickle cell disease activates peripheral blood mononuclear cells to induce cathepsins k and v activity in endothelial cells.</a> Anemia. 2012, 201781 (2012).</li><li>Allison, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+sickle-cell+haemoglobin.">Properties of sickle-cell haemoglobin.</a> Biochemical Journal. 65, 212 (1957).</li><li>Kuehl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design of experiments : statistical principles of research design and analysis. , (2000).</li><li>Lindman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of variance in experimental designs. , (1992).</li><li>Pan, W., Galkin, O., Filobelo, L., Nagel, R. L., Vekilov, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastable+Mesoscopic+Clusters+in+Solutions+of+Sickle-Cell+Hemoglobin.">Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin.</a> Biophysical Journal. 92, 267-277 (2007).</li><li>Wootton, D. M., Popel, A. S., Alevriadou, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+theoretical+study+on+the+dissolution+of+mural+fibrin+clots+by+tissue-type+plasminogen+activator.">An experimental and theoretical study on the dissolution of mural fibrin clots by tissue-type plasminogen activator.</a> Biotechnology and bioengineering. 77, 405-419 (2002).</li><li>Sazonova, I. Y., 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=127+Mathematic+model+of+fibrin+clot+lysis+by+plasmin.">127 Mathematic model of fibrin clot lysis by plasmin.</a> Fibrinolysis and Proteolysis. 12, 45 (1998).</li></ol>

The authors have nothing to disclose.

To obtain meaningful data about the structure of clotting mechanisms in disease states, it is important to isolate the factors involved in clotting to determine the effects of the proteins and cells in these conditions. This protocol was developed for the purposes of investigating the structure of the fibrin clot in diabetic and SCD states in vitro.
It is necessary to understand the mechanisms involved in fibrin formation and fibrinolysis in disease states since altered conditions cau...

Injury to a blood vessel&#39;s endothelial lining is repaired through the hemostatic response, or the formation of a blood clot. When blood permeates into the extracellular matrix, tissue factors activate platelets in the blood stream that facilitate initiation of the coagulation cascade. The key mechanical component of this healing process is the fibrin matrix, composed of fibrin fibers that are highly elastic, and can sustain large forces 1-4. Many researchers have studied the formation structure, and function of fibrin extensively in the past decades 5-13.
Patients with diseases such as diabetes mellitus and sickle cell have an increased risk of developing thrombotic complications such as myocardial infarctions, ischemic heart disease, and strokes 14-19. Over 2 million people are newly diagnosed with diabetes mellitus each year in the United States. There are two types of diabetes: Type I, where the body fails to produce adequate amounts of insulin, and Type II, where the body becomes resistant to insulin. Among diabetic patients, cardiovascular disease (CVD) is the cause for 80% of the morbidity and mortality associated with the disease 20,21.
Sickle cell disease (SCD) is a genetic blood disorder that affects more than 100,000 people in the United States22. SCD is a point-mutation disease that causes red blood cells to become crescent-shaped, making it difficult for the cells to pass through the blood vasculature23. Both of these disease states increase the chances of developing atherothrombotic conditions in the body. One of the reasons for this is a result of altered fibrin structure and function in diseased states14,24-26.
In both diabetes and sickle cell disease, there is hypercoagulation and hypofibrinolysis activity that induces atherothrombosis and cardiovascular disease (CVD) as compared to healthy patients 17,27,28. It is known that hypofibrinolysis promotes atherosclerosis progression and engenders recurrent ischemic events for patients with premature coronary artery disease29. In the current manuscript, we investigated the role of fibrin physical properties in this particular setting. Fibrin clot structures in non-diseased patients are composed of thin fibers, larger pores, and generally less dense14,24. The increased porosity and less dense fibrin clots in healthy patients have been found to facilitate fibrinolysis16. In hyperthrombotic conditions such as diabetic and sickle cell disease, there is an increase in fibrinogen production, causing the fibrinogen concentration to increase from normal levels of 2.5 mg/ml in healthy patients 30-33. Fibrin clots formed in diabetic patients have been found to be less porous, more rigid, have more branch points, and denser when compared to healthy, non-diabetic patients14,24,33-35. The altered fibrin structure is a result of glycation mechanisms that occur in the proteins involved in clot formation. Nonenzymatic (irreversible) glycation occurs when glucose molecules bind to lysine residues on the fibrinogen molecule, which inhibits human factor XIIIa (FXIIIa) from properly cross-linking glutamine and lysine residues33,36,37.
The structural analysis of fibrin networks has been studied extensively recently. In particular, researchers have utilized electron microscopy and 3D reconstruction of fibrin networks38, investigated how both intravascular (endothelial) cells and extravascular (fibroblasts and smooth muscle) cells affect fibrin structure39, utilized viscoelastic and spectral analysis to analyze fibrin structures40 , and developed correlations between fibrin structure and mechanical properties using experimental and computational approaches41. The focus of the current study was to formulate clot structures under simulated diabetic and sickle cell thrombosis conditions and to use confocal microscopy for examination of the structure and function of the clots in diseased states. Fibrin clots were formed from human fibrinogen, human thrombin, and FXIIIa. The clots were lysed using plasmin. To simulate diabetic conditions, increased concentration of fibrinogen was incubated in glucose solution to induce in vitro fibrinogen glycation. To simulate sickle cell disease clotting conditions, increased fibrinogen concentrations were mixed with sickle cell hematocrit collected from patients as done previously by our group42. These methods were used to examine the structure and functions involved in fibrin clot formation and fibrinolysis under diseased conditions, as well as the mechanisms that induce CVD. Based on current information about these diseases, the glycated fibrin clot structures were denser with fewer and smaller pores. The fibrin clots with red blood cells from sickle cell patients (RBCs) were also denser and displayed aggregation of the RBCs and agglomerated fibrin clusters. This is a well-established phenomenon that has been determined previously43. It was also hypothesized that the fibrinolysis rate would be significantly lower in glycated fibrin clots with and without reduced plasmin compared to healthy, normal fibrin. The results showed that for glycated fibrin clots, significantly different lysis rate results were observed only under conditions of reduced plasmin concentration. This experimental technique of using confocal microscopy offers significant advantages over other imaging methods because the cells and proteins remain in their native state, which enables the capturing of real-time video of the clotting activity. This method of synthetically inducing clotting is also cheaper and more time efficient than obtaining patient samples and filtering out individual proteins and enzymes. Furthermore, by using separated proteins and enzymes to synthesize clots, the clots were standardized so that there was not variability between samples as a result of other proteins in the plasma.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">PBS</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ficoll-Paque (hydrophilic polysaccharide)</td>
			<td style="width:287px;">GE Healthcare</td>
			<td style="width:119px;">45-001-749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml heparinized vacutainer tubes</td>
			<td style="width:287px;">BD Biosciences</td>
			<td style="width:119px;">366643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Human Fibrinogen</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 human fibrinogen conjugate</td>
			<td style="width:287px;">Molecular Probes</td>
			<td style="width:119px;">F13191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mL graduated microcentrifuge tube</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Glucose powder</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">15023-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">FXIIIa</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VIS Confocal Microscope</td>
			<td style="width:287px;">Zeiss LSM 510</td>
			<td style="width:119px;">LSM 510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50 mM Tris</td>
			<td style="width:287px;">Lonza</td>
			<td style="width:119px;">S50-642</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:315px;">Calcium Chloride (CaCl2)</td>
			<td style="width:287px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiD cell-labeling solution</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">L7781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Plasmin</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium Chloride (5 M NaCl)</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">AM9759</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Statistical Modeling Software</td>
			<td>IBM</td>
			<td style="width:119px;">SPSS Statistics 22</td>
			<td></td>
		</tr>
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experimental and imaging techniques for examining fibrin clot structures in normal and diseased states

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

Confocal Microscopy Analysis of Glycated Fibrin Clot Structures
The confocal microscopy images of normal and glycated clots are presented in Figure 3. Confocal microscopy analysis of the normal and glycated clots reveals that glycated clots are denser with smaller pores than the normal clots both with and without the addition of FXIIIa during clot polymerization. In Figure 3A and 3B, there is a lower fibrin concentration which created a less d...

Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

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11.3K Views.  Georgia Institute of Technology & Emory University School of Medicine. In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Video: Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

&lt;em&gt;In Vivo&lt;/em&gt; Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&amp;#232;re&#039;s Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...