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><tbody><tr><td>Ethilon P-3 13 mm 3/8 circle 5-0</td><td>Ethicon, Norderstedt, Germany</td><td>698G</td><td>Ethilon polyamid-6 precision point-reverse cutting suture</td></tr><tr><td>Biopsy punches (10 mm)</td><td>Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td><td>P1050</td><td></td></tr><tr><td>Biopsy punches (12 mm)</td><td>Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td><td>P1250</td><td></td></tr><tr><td>Digital camera&amp;nbsp;</td><td>Ricoh, Hannover, Germany</td><td>Cx1</td><td></td></tr><tr><td>Gazin Mullkompresse&amp;nbsp;</td><td>Lohmann und Rauscher, Neuwied, Germany</td><td>13622</td><td>Sterile gauze (10 cm x 10 cm)</td></tr><tr><td>Double-layer collagen-based scaffold (8&#039; x 10&#039;)</td><td>Integra Life Science Corporation, Plainsboro, NJ, USA</td><td>88101</td><td></td></tr><tr><td>Isoflurane, liquid-gas for inhalative anesthesia&amp;nbsp;</td><td>Baxter, Unterschleissheim, Germany</td><td>100196040</td><td></td></tr><tr><td>Pentobarbital, 16 g / 100 ml</td><td>Fa. Merial, Hallbergmoos</td><td></td><td></td></tr><tr><td>Nuri Nu/Nu Nude mice, CrLNU-Foxn1&lt;sup&gt;nu&lt;/sup&gt;</td><td>Charles River, Sulzfeld, Germany</td><td>Strain code 088</td><td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&amp;nbsp;</td></tr><tr><td>Buprenorphine (0.3 mg/ml)</td><td>Essex Pharma GmbH, Munich, Germany</td><td></td><td></td></tr><tr><td>Titanized mesh (15 cm x 15 cm), extralight</td><td>PFM Medical AG, K&amp;ouml;ln, Germany</td><td>6000029</td><td></td></tr><tr><td>Tissucol Duo S Immuno 2 ml</td><td>Baxter Germany GmbH, Unterschlei&amp;szlig;heim, Germany</td><td>B1332020110614</td><td>Fibrin-thrombin solution&amp;nbsp;</td></tr><tr><td>Transparent adhesive drape (30.5 cm x 26 cm)</td><td>KCI Medical Products, Wimborne Dorset, UK</td><td>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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Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.1K Views.  Technische Universität München. 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. 

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

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 ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

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 ...

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Cancer Research

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

Developmental Biology

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

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

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

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

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

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Biology

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

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

Summary

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Chapters in this video

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

Engineered Vascularized Muscle Flap

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

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A Patient-Derived Xenograft Model for Venous Malformation

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

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels