Integra dermal regeneration template was kindly provided by Integra LifeSciences Corporation. Sources of funds supporting the work: This work was partially financed by the CIRM-BMBF Early Translational II Award and the FONDAP Center for Genome regulation (Nr. 15090007) both to JTE.

1. Preparation of Scaffolds
<ol>
	<li>Generate samples of the scaffolds by using 12 mm biopsy punches.</li>
	<li>To introduce bioactive molecules or cells into the scaffold, drain the scaffolds by gently squeezing them with a sterile gauze. Then rehydrate the scaffolds by adding 160 &#181;l of a solution containing the bioactive molecules or cells of interest. Double-check the success of bioactivation with cells through metabolic assays such as MTT Assays.</li>
	<li>If necessary, fix the compounds or cells to be tested inside the scaffold by delivering them, for instance in a fibrin-thrombin solution or a hydrogel, again through rehydration as described in step 1.2. 
	NOTE: This step can be of helpful when compounds or cells do not mechanically attach to the material. One can also control the release dynamics of the compounds..</li>
	<li>Prepare the titanized meshes, which will be placed under each scaffold in every experiment and control group, by cutting out round shaped pieces, approximately 14 mm in diameter. 
	NOTE: The titanized mesh works as a physical border required to delimit the regenerated tissue from the wound bed.</li>
</ol>
2. Animals
<ol>
	<li>Prior to implementation of the presented model, consult the corresponding animal protection laws and obtain permission from the local authorities. In this work, all experiments were performed with mice, according to the current German animal welfare act and approved by the District Government of Upper Bavaria (Regierung von Oberbayern).</li>
	<li>Carefully choose the animal and strain. Although the model was established for mice, it can be translated to other common animals, such as rats, rabbits or pigs.</li>
	<li>In case you are working with furred animals, shave the back of the animals before implantation of the scaffolds. 
	NOTE: This work uses mice of 6 to 8 weeks of age with a body weight between 20 g and 25 g, however different ages and body weights can also be used.</li>
</ol>
3. Anesthesia
<ol>
	<li>Conduct the operation under sterile conditions. In order to maintain the animal&#8217;s normal body temperature, use a warming mat.</li>
	<li>Prior to inhalative anesthesia the animals receive Buprenorphin at a dose of 0.05-0.1 mg/kg bodyweight, subcutaneously every 8 hr.</li>
	<li>Place the animal under inhalative anesthesia (Isoflurane) or apply equivalent standard procedures. Confirm anesthesia either by gently squeezing the animal&#8217;s toes or through a skin pinch.</li>
	<li>To prevent exsiccosis, inject 0.5 ml physiologic saline solution subcutaneously at the beginning of the operation.</li>
</ol>
4. Excision of the Skin
<ol>
	<li>Place the animal in a prone position and mark the midline of the mouse&#8217;s back with a fine tip permanent pen (Figure&#160;1A).</li>
	<li>Define the excision area. Place the defects in the area shown in Figure 1C. Note that if the defects are placed too caudally, animals are more prone to remove the dressings and the scaffolds.</li>
	<li>Create round bilateral defects using a 10 mm biopsy punch (Figure&#160;1B). The punch should be carefully forced against the skin and must not be used to cut completely through the skin, but to delineate the excision area (Figures&#160;1C &#38; 1E).</li>
	<li>Gently lift off the marked skin with forceps and incise along the marked circle using fine surgical scissors (Figure&#160;1F). In case of bleeding, carefully compress with sterile gauze. A step-by-step description of the excision is shown in Figure 1. 
	NOTE: The defect is created with a smaller punch than the one for generating the scaffolds, to compensate the enlargement of the defect due to the skin elasticity.</li>
</ol>
5. Scaffold Implantation
<ol>
	<li>In order to make space for the titanized mesh, extend the separation of skin and underlying tissue at the wound borders for 2-4 mm (Figure&#160;2A).</li>
	<li>Place the titanized mesh into the defect directly on the wound bed and under the wound edges (Figures&#160;2A &#38; B).</li>
	<li>Place scaffolds directly over the mesh.</li>
	<li>Suture the scaffolds to the adjacent wound edges with 4 to 6 single knots, leaving the edges slightly over the scaffold (Figure&#160;2C). 
	NOTE: Avoid the use of biodegradable sutures.</li>
	<li>Suture a transparent wound dressing above the defects to protect the scaffold while allowing monitoring of the wound area (Figure&#160;2D).</li>
</ol>
6. Postoperative Care
<ol>
	<li>Keep one mouse per cage to avert mutual manipulation of the dressing and scaffolds.</li>
	<li>Evaluate the general state of the animal on a daily basis by observing motor activity, body weight, signs of pain, tolerance to the dressing and auto-mutilation. Also monitor the wound area for bleeding, local and systemic signs of infection and the position of the dressing.</li>
	<li>In order to minimize the pain of the animal, inject Buprenorphin daily at a dose of 0.05-0.1 mg/kg bodyweight or equivalent analgesic medication.</li>
	<li>If the mouse removes or damages the wound dressing, replace or reattach it under anesthesia.</li>
</ol>
7. Euthanasia and Explantation of the Scaffold
<ol>
	<li>At desired time points, sacrifice the animals by overdoses of Pentobarbital (150 mg/kg).</li>
	<li>Mark the site of skin excision with a permanent marker, which should include the scaffolds and as much of the mouse back skin as possible (Figure&#160;3A).</li>
	<li>Incise the skin along the marked lines with a pair of scissors or a scalpel. Detach the entire skin, including the scaffolds and the titanized mesh, from the underlying tissue through blunt preparation (Figure&#160;3B).</li>
	<li>Place the explanted tissue stretched out on a Petri dish (Figure&#160;3C and D)</li>
</ol>
8. Visualization and Quantification of the Vascular Network
<ol>
	<li>Transillumination:
	<ol>
		<li>Set up a transillumination device, by mounting a Petri dish above a strong source of white light (100 Watt standard light bulb).</li>
		<li>Fix a digital camera above the transilluminator to obtain digital pictures.</li>
		<li>Place the samples upside down on the Petri dish over the transillumination device.</li>
		<li>Take pictures of the complete scaffolds in macro mode as well as of equally sized areas of normal skin. Store the images in a TIFF format for further digital analysis.</li>
		<li>Save the tissue for further biochemical or histological analysis.</li>
	</ol>
	</li>
	<li>Digital segmentation and quantification:
	<ol>
		<li>Download and install the VesSeg-Tool software, which can be obtained free of charge at: <a href="http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255">http://www.isip.uni-luebeck.de/index.php?id=150&#38;L=2%255</a>.</li>
		<li>Open the picture with the VesSeg-Tool software.</li>
		<li>Select the area of the image covered by the scaffold for digital analysis (Image&#8594;Select).</li>
		<li>To increase the contrast of vessels use the option &#8220;Hysteresis thresholding&#8221; (Image&#8594;Vessel enhancement filter&#8594;Hysteresis thresholding&#8594;Top-Hat transformation&#8594;Calculate vessel enhancement).</li>
		<li>Proceed with the segmentation of the vessel map (Image&#8594;Vessel enhancement filter&#8594;Hysteresis thresholding&#8594;Threshold borders). Select the first threshold (&#8220;Vessel coverage&#8221;), so every pixel, which is even remotely vessel-like, will be labeled as vessel. Select the second threshold (&#8220;Background coverage&#8221;), so only those pixels that are particularly vessel-like will be labeled as vessels.</li>
		<li>Check the automatic segmentation proposal manually to add non-captured vessels and to eliminate common false-positive structures, which usually are long and thin, vessel-like structures such as hairs or sutures.</li>
		<li>Calculate the length of the vessels and the overall area covered by vessels (Image&#8594;Image statistics&#8594;Binary image statistics). 
		NOTE: For the calculation of the vessel-lengths, vessels in the resulting vessel map are thinned to lines with a width of one pixel20.</li>
	</ol>
	</li>
	<li>Analyze the native skin area under the same parameters as the scaffolds, assigning a value of 100% to the native tissue and relate the scaffold to that value. For instance, if the percentage of white pixels is 60% in the normal tissue and 30% in the scaffold, it would represent a 50% vascularization of the scaffold. Note: The white vessels coverage is assigned to the area of a square around the scaffold. Adjust the number of white pixels coverage considering that the area of the circle is significantly smaller (&#960; x R2) than the square (4 x R2).</li>
</ol>

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Conflict of interest statement:
All authors: None
Financial Disclosures:
None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript.

There is a need to establish successful approaches in improving blood perfusion in tissue engineered constructs, which demands the development of new reliable methods to study the vascularization processes within the biomaterials. Common methods for making scaffold vascularization ex vivo visible include the use of microscopy, which provides a high-resolution tool. In most cases, though, this method is limited to the analysis of small tissue areas and tends to be expensive and time consuming. Moreover, it often ...

In the recent decades, tissue engineering has opened up a new therapeutic option to replace tissue defects with the body&#8217;s own cells1. In order to support the physiological process of tissue regeneration, scaffolds are designed as a biodegradable structure, that provides a scenario where cells from the wound bed can grow and restore the defect2,3.
Insufficient vascularization is considered to be the main obstacle, which holds back the clinical breakthrough of bioartificial scaffolds4. With the ingrowth of cells, the demand for nutrients and oxygen increases and vascularization of the material becomes essential. Insufficient or delayed vascularization can therefore lead to central necrosis of tissue-engineered products5. In addition, blood vessels provide immune competent cells and remove the metabolic residues in the regenerating area. High infection rates and low regeneration are only some of the consequences of insufficient blood perfusion observed in tissue engineering, which are aimed to be averted by increasing the vascularization of the scaffolds6,7.
Several strategies that aim at improving vascularization focus on the key role of the biomaterial itself and the microstructure of the scaffold. There are intensive research efforts to develop new approaches in shifting the healing process from repair to regeneration, thereby (re)generating a tissue with the closest physiological properties to the one to be restored8,9. Biomaterials that were studied and evaluated with regards to their regenerative potential included collagen, fibrin, chitosan and alginate10,11. These biomaterials can be used and combined as a backbone for building new scaffolds using different strategies such as tissue decellularization, self-assembly, rapid prototyping and electrospinning12. In order to enhance the body&#8217;s own regenerative capacity, scaffolds can be bioactivated. The incorporation of recombinant angiogenic growth factors13 or gene vectors encoding for such factors14 has shown to improve vascularization of the scaffold. The use of stem cells has been widely shown to be a promising strategy to improve vascularization, where mesenchymal stromal cells and endothelial progenitor cells have gained the most attention15,16. Other approaches attempt to build constructs that contain prefabricated vessel networks prior to transplantation17. Despite intensive efforts in scaffold design and their bio-activation, no strategy has improved vascularization at a clinically significant level and, with the exception of dermal replacements in massive burn injuries, the translation of bioengineered materials into the clinical routine is only taking place hesitantly18.
One of the reasons why vascularization of artificial tissue constructs is still an unsolved problem, is the difficulty to evaluate the success of new technologies in in vivo approaches. Although in vitro experiments may provide important insights of the vascularization potential of scaffolds, appropriate animal models are required to study key parameters such as the biocompatibility of the material, the safety and efficacy of the treatment and, of particular importance, the vascularization of the tissue construct. Therefore, reliable tools to visualize and quantify blood vessel networks in vivo are essential.
In this study we present a simple and reliable method that allows the visualization and quantification of the vascular network inside explanted scaffolds. This method is based on tissue transillumination and digital segmentation. Since this method is non-invasive, it allows further molecular and histological analyses of the target material.

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name of Material/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:431px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Ethilon P-3 13 mm 3/8 circle 5-0</td>
			<td style="width:223px;">Ethicon, Norderstedt, Germany</td>
			<td style="width:119px;">698G</td>
			<td>Ethilon polyamid-6 precision point-reverse cutting suture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (10 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (12 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital camera&#160;</td>
			<td style="width:223px;">Ricoh, Hannover, Germany</td>
			<td style="width:119px;">Cx1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gazin Mullkompresse&#160;</td>
			<td style="width:223px;">Lohmann und Rauscher, Neuwied, Germany</td>
			<td style="width:119px;">13622</td>
			<td>Sterile gauze (10 cm x 10 cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Double-layer collagen-based scaffold (8 &#39;x 10&#39;)</td>
			<td style="width:223px;">Integra Life Science Corporation, Plainsboro, NJ, USA</td>
			<td style="width:119px;">88101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, liquid-gas for inhalative anesthesia&#160;</td>
			<td style="width:223px;">Baxter, Unterschleissheim, Germany</td>
			<td style="width:119px;">100196040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pentobarbital, 16 g / 100 ml</td>
			<td style="width:223px;">Fa. Merial, Hallbergmoos</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu</td>
			<td style="width:223px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:119px;">Strain code 088</td>
			<td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Buprenorphine (0.3 mg/ml)</td>
			<td style="width:223px;">Essex Pharma GmbH, Munich, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Titanized mesh (15 cm x 15 cm), extralight</td>
			<td style="width:223px;">PFM Medical AG, K&#246;ln, Germany</td>
			<td style="width:119px;">6000029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Tissucol Duo S Immuno 2 ml</td>
			<td style="width:223px;">Baxter Germany GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;">B1332020110614</td>
			<td>Fibrin-thrombin solution&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Transparent adhesove drape (30.5 cm x 26 cm)</td>
			<td style="width:223px;">KCI Medical Products, Wimborne Dorset, UK</td>
			<td style="width:119px;">M6275009/10</td>
			<td></td>
		</tr>
	</tbody>
</table>

a full skin defect model to evaluate vascularization of biomaterials in vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A reliable bilateral full skin defect can be created in the mouse (Figure&#160;1) where the skin can be replaced by a biomaterial under study (Figure&#160;2). Here, no major complications are observed during or after the operative procedure, neither macroscopic signs of infection or foreign body reaction. In rare cases, a scaffold gets lost when a mouse removes it. Wound contraction was never observed (Figure&#160;3). Tissue transillumination allowed clear visualization ...

Watch this Scientific Journal Video about A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo at JoVE.com

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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JoVE Journal

Bioengineering

12.1K vistas.  Technische Universität München.  La vascularización es clave para enfoques en la ingeniería de tejidos éxito. Por lo tanto, se requieren tecnologías fiables para evaluar el desarrollo de las redes vasculares en los tejidos-construcciones. Aquí presentamos un método simple y rentable para visualizar y cuantificar la vascularización in vivo. 

Video: A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...