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.
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’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.
In the recent decades, tissue engineering has opened up a new therapeutic option to replace tissue defects with the body’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’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.
1. Preparation of Scaffolds
2. Animals
3. Anesthesia
4. Excision of the Skin
5. Scaffold Implantation
6. Postoperative Care
7. Euthanasia and Explantation of the Scaffold
8. Visualization and Quantification of the Vascular Network
A reliable bilateral full skin defect can be created in the mouse (Figure 1) where the skin can be replaced by a biomaterial under study (Figure 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 3). Tissue transillumination allowed clear visualization of vascular structures of up to 30 μm in width, in a whole tissue sample that included both, native tissue and implanted scaffolds (Figure 4). After digital segmentation, vessels could be easily quantified at desired times post implantation. For instance, 2 weeks after implantation, vascularization levels of 62.28 ± 8.6% were observed (mean ± standard error of mean; n=8)21. Altogether, about 25 min per animal (2 scaffolds) are required for the implantation, 5 min for tissue harvesting and picturing and 10 min for digital analysis of one sample. As a final point, this method does not affect the quality of the explanted tissue for further analysis. For instance, proteins and RNA extracts can be easily obtained from the sample by standard methods.
Figure 1. Full Skin Defect. The midline of the mouse’s back is marked with a fine tip pen and a biopsy punch is used to mark the area were the defect will be created (A, B, C and D). Once the areas are defined, full skin is removed with standard surgical scissors as seen in the close-up of the preparation (E and F). A final result of the defect is shown in (D) and magnified in (G). Scale bars represent 1 cm in ABCD and 5 mm in EFG. Please click here to view a larger version of this figure.
Figure 2. Implantation of the scaffold. Prior to the implantation of the scaffold, a mesh is placed into the defect directly on the wound bed and under the wound edges (A and B). Next scaffolds are placed over the mesh and sutured to the wound edges (C). Finally, a transparent wound dressing is placed and sutured to the skin (D). Scale bar represents 5 mm in (A, B, and C) and 1 cm in (D). Please click here to view a larger version of this figure.
Figure 3. Tissue explantation. For tissue analysis the full skin from the back of the animal is surgically removed (A and B) and stretched out on a Petri dish (C). For transillumination the skin is turned upside down and a digital picture is taken (D). Removal of the skin next to the scaffolds is necessary for normalization of the vascularization data. Scale bars represent 1 cm. Please click here to view a larger version of this figure.
Figure 4. Transillumination, digital segmentation and quantification. Skin tissue was excised and the vascular network within was visualized by transillumination (upper pictures) Full skin of the back (A) and detailed areas of the native skin (B) and the scaffold (C) are shown. Arrows show the magnified areas in (B) and (C). Each of the lower pictures shows the results of the digital segmentation process for the picture located directly above. Scale bars represent 5 mm. Please click here to view a larger version of this figure.
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 includes elaborate labeling and staining methods, where blood perfused vessels cannot be distinguished from non-functional ones22. Methods in use to assess the functionality of vessels are computer tomography, magnetic resonance imaging and positron electron tomography. These methods have the drawbacks of extremely costly equipment and low-resolution power23. Another commonly used strategy to visualize vascularization is the use of vascular casts followed by chemical corrosion of the tissue24. This method makes it possible to obtain a representative 3D structure of the vascular network, but the results strongly vary depending on the level of artificial perfusion and tissues cannot be used for further biochemical studies, such as protein or RNA analysis. The chicken chorioallantoic membrane (CAM) model25 and the skinfold chamber model in rodents26,27 have shown to be effective methods to analyze angiogenesis, capillary perfusion and blood flow. Recently, the skinfold chamber model has also been used for tissue engineering applications evaluating MSC-based vascularization strategies in porous polyurethane scaffolds28. These methods present important advantages for in vivo imaging and represent an attractive target for the method described here.
The model presented here was developed to overcome the shortcomings of the methods mentioned above. Taking advantage of the unique intrinsic characteristics of the skin, full defects are created and used to evaluate vascularization of biomaterials. Skin is an external organ, which allows easier visualization and manipulation of the tissue. Because of its large surface and homogeneous structure, multiple defects can be created which make internal comparisons possible, while decreasing the number of animals required for the study. Due to the antero-posterior symmetry of the animal, bilateral defects are suggested, however; the influence of systemic effects should be considered when both sides are compared to each other. Finally, skin is a fairly transparent tissue representing an optimal organ of choice for transillumination. Although the method described here is suited for many different native tissues and several biomaterials, this protocol was originally established to be used with a double-layer collagen-based scaffold that is FDA-approved and clinically used for dermal repair. An important prerequisite for the implementation of this method is that the scaffold has to be partially transparent. Depending on the chosen scaffold material, some adaptation of the protocol might be required. Previously, our group has used the model to show how the application of human mesenchymal cells to scaffolds enhances their vascularization29.
A standard digital camera and a light source are sufficient for the visualization of the vascular network, which can be quantified through free software. The semiautomatic segmentation of the pictures by the VesSeg-Tool consists of two steps. In the first step, the image taken from the vascular network is contrast-enhanced leading to a vessel map. Then, the segmentation algorithm selects the vessel areas and finally, the segmentation proposal is presented as an image (Figure 4)30. In a second processing step, using the fact that vessels are connected structures (meaning that vessel pixels hang together spatially), a second segmentation is used to select from the first one only the true vessel pixels and all true vessel pixels. Therefore, a final label of “one” is awarded only to those low-confidence vessel pixels from the first image that are connected over a chain of neighbors to a high-confidence vessel pixel in the second image.
Since no chemical additives are necessary, this method is also cost-effective. Digital analysis shows a good reproducibility at determining vessel dimensions, intravascular distances, number of branching points, vascularized area and total length of vascular network. We have previously shown that the blood vessel network can be better visualized by transillumination than through radio-angiographic studies31. The presence of the titan mesh has a double function. On the one hand, it prevents wound contraction, which is a common drawback of the use of mouse models; on the other hand, it provides a physical border between the scaffold and the wound bed. Because most scaffolds are biodegradable, the last-mentioned function is critical to physically delimitate the border between the scaffold and the wound bed during the harvesting of the scaffolds. Among the main drawbacks of this method is the need of explanting the scaffolds and therefore the restrain of the analysis to one single time point; thus the dynamic observation of vascularization is not possible with the current setting. Another problem is that this method is limited to semi-transparent biomaterials that permit transillumination. Finally, even though the operation procedure does not require special surgical skills, a learning curve of about 6 – 12 operated animals have to be considered to master the method properly.
We introduce a time and cost effective approach that allows high-resolution visualization and quantification of vascular structures, representing an ideal method to compare vascularization in different biomaterials or different scaffold-bioactivation strategies. This method is easy to establish and does not require special equipment. A key feature of this model is that after vessel visualization, tissues can be further used for studies such as histological or molecular analysis.
The authors have nothing to disclose.
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.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Ethilon P-3 13 mm 3/8 circle 5-0 | Ethicon, Norderstedt, Germany | 698G | Ethilon polyamid-6 precision point-reverse cutting suture |
Biopsy punches (10 mm) | Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA | P1050 | |
Biopsy punches (12 mm) | Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA | P1250 | |
Digital camera | Ricoh, Hannover, Germany | Cx1 | |
Gazin Mullkompresse | Lohmann und Rauscher, Neuwied, Germany | 13622 | Sterile gauze (10 cm x 10 cm) |
Double-layer collagen-based scaffold (8 'x 10') | Integra Life Science Corporation, Plainsboro, NJ, USA | 88101 | |
Isoflurane, liquid-gas for inhalative anesthesia | Baxter, Unterschleissheim, Germany | 100196040 | |
Pentobarbital, 16 g / 100 ml | Fa. Merial, Hallbergmoos | ||
Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu | Charles River, Sulzfeld, Germany | Strain code 088 | Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g |
Buprenorphine (0.3 mg/ml) | Essex Pharma GmbH, Munich, Germany | ||
Titanized mesh (15 cm x 15 cm), extralight | PFM Medical AG, Köln, Germany | 6000029 | |
Tissucol Duo S Immuno 2 ml | Baxter Germany GmbH, Unterschleißheim, Germany | B1332020110614 | Fibrin-thrombin solution |
Transparent adhesove drape (30.5 cm x 26 cm) | KCI Medical Products, Wimborne Dorset, UK | M6275009/10 |