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 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.
Fabricating functional vascularized tissue using a tissue engineering approach is an emerging paradigm in regenerative medicine.1,2 Many approaches to engineer new and healthy tissue for the replacement of injured tissue or defective organs have been developed,3-6 experimentally in small animal models with promising clinical potential.7,8 However, vascularization remains one of the great challenges for tissue engineering limiting its potential to grow tissues of clinically relevant size.9
Current approaches to vascularize tissue follow either an extrinsic pathway where new vessels grow from the recipient vascular bed and invade throughout the implanted tissue constructs10 or an intrinsic vascularization pathway where the vasculature grows and expands in unison with the newly developing tissue.11 The extrinsic approach traditionally involves seeding cells onto a scaffold in vitro and implanting the complete construct into the living animal with the expectation that nutrients, previously supplied by culture media, will be sourced from the circulation.12,13 The concept is simplistic as vascular ingrowth is too slow and only very thin implants (<1-2 mm thick) will remain viable. Providing nutrients and oxygen by means of a sufficient and rapid vascularization is at the heart of any successful attempts to grow more complex and larger tissue-engineered substitutes such as bone, muscle, fat and solid organs.14,15 Intrinsic vascularization offers the potential for larger constructs to develop by progressive tissue growth commensurate with its expanding blood supply. One design is the in vivo implantation into a chamber of a vascular pedicle with or without a cell seeded scaffold.5,6 This has paved the way to new procedures for the generation of thicker intrinsically vascularized tissues.16,17
More recently, strategies have been developed to pre-vascularize tissue grafts, prior to implantation. These incorporated blood vessel networks are aimed to inosculate with host vessels at implantation allowing for the rapid provision of a complete blood supply to improve the survival of all parts of a transplanted thick tissue graft.18
We pioneered an in vivo vascularized tissue engineering model in small animals that involves a subcutaneously implanted semi-rigid enclosed chamber containing a perfused vascular pedicle and cell-containing biomaterials. The chamber creates an ischemic environment that stimulates angiogenic sprouting from the implanted vessels.3 The vascular pedicle can either be a reconstructed arteriovenous loop or an intact flow-through artery and vein.3-6,19 This vascular pedicle sprouts a functioning and extensive arterio-capillary-venous network that links at both arteriole and venous ends with the vascular pedicle.3,20 Furthermore, the surrounding hollow support chamber protects the developing tissue from potentially deforming mechanical forces and prolongs the ischaemic drive to enhance vascularization.3,21,22 If the vessel pedicle is simply implanted into normal tissue and not inside the protected space of the chamber, angiogenic sprouting ceases along the same timeline as a normal wound and no new tissue will accumulate around the pedicle. Investigators have used this in vivo configuration to produce three-dimensional functional vascularized tissue constructs with supportive vasculature and of clinically relevant size.4,23 Furthermore, the engineered vascularized tissue constructs with its intact vascular pedicle can be harvested for subsequent transplantation at the injury site.24,25 A more clinically feasible scenario would be creating the chamber at the definitive site for reconstruction such as the breast. Thus, this de novo tissue engineering approach could have clinical potential to provide a new source of functional target tissue for reconstructive surgery.26-28
The following protocol will provide a general guide to construct an in vivo vascularized tissue engineering chamber in the rat, which could be adapted in different animal models and employed to examine the intricate processes of angiogenesis, matrix production, and cellular migration and differentiation.
The protocols described here have been approved by the Animal Ethics Committee of St. Vincent's Hospital Melbourne, Australia, and were conducted under strict adherence to the Australian National Health and Medical Research Council Guidelines.
NOTE: Two chamber protocols are described below. The two different models and their specific chamber designs are illustrated in Figure 1. Chamber (1) is made of polycarbonate (for rat arteriovenous loop chamber model). It is cylindrical with an internal diameter 13 mm and height 4 mm. A window at one point in the wall allows unimpeded access for the pedicle. In the second model (for rat flow-through pedicle chamber model), the chamber is made of polyacrylic and is rectangular (10 x 8 x 4 mm3 internal dimensions). It has two 1.5 mm openings on opposite sides to accommodate the femoral artery and vein as they transgress the chamber.
1. Rat Arteriovenous Loop Chamber Model (One Chamber Per Animal)
NOTE: Prior to starting surgery, make sure all the instruments have been properly sterilized. Likewise, ensure the instruments rest on sterile towels and are at a reasonable distance from the surgical field to avoid contamination during the procedure.
2. Flow-through Pedicle Chamber (Two Chambers per Animal)
3. Harvest of Chambers and Tissue Processing
The microsurgical creation of tissue engineering chambers was performed as described in the protocol above. Tissues generated inside the chambers can be examined histologically as describe in protocol step 3. Various tissue types have been successfully engineered using the in vivo vascularized chamber (Figure 2). These include cardiac tissue with neonatal rat cardiomyocytes (Figure 2A), muscle tissue with rat skeletal myoblasts (Figure 2B), and adipose tissue with a hydrogel derived from adipose tissue extracellular matrix (Figure 2C).
Morphometric evaluation of the tissues can be performed with either a stereo investigator system or ImageJ software.3,4,8,17,22,29 In addition to qualitative assessment of various tissue components, the stereology system also allows for unbiased quantification of specific tissue volume. For example, lectin (a marker for rodent endothelial cells) stained transverse sections (Figure 3) can be used to estimate the vascular volume of the harvested tissue constructs using video microscopy with stereology system. Similar quantification methods can be applied to assess the volume of other tissue types.
The tissue engineering chambers can also be employed to track cell fate following in vivo implantation. Cells can be pre-labelled with fluorescent dyes such as DiI, PKH26 or quantum dots before implantation. For example, neonatal rat cardiomyocytes pre-labelled with DiI can be detected in the tissue constructs harvested at 3 day post-implantation (Figure 4A). We have also successfully tracked implanted cells that have been pre-labelled with DiI for up to 4 weeks post-implantation. Alternatively, species-specific antibodies can be used to identify implanted cells in xenotransplantation studies. For example, human induced pluripotent stem cells implanted inside tissue engineering chambers in immunocompromized rats can be identified in the harvested tissue constructs by immunostaining with human-specific Ku80 antibody (Figure 4B).
Figure 1: Cardiac tissue engineering with the in vivo vascularized chamber. Intrinsic vascularization approach with arteriovenous loop chamber model and flow-through pedicle chamber model. The chambers were made from either polycarbonate or polyacrylic, these materials were tested to be non-inflammatory and non-toxic in vivo. Scale bar = 5 mm. Re-printed with permission from30. Please click here to view a larger version of this figure.
Figure 2: Tissues engineered from the in vivo vascularized chambers. (A) Cardiac tissue with neonatal rat cardiomyocytes. Cardiomyocytes were immunostained with cardiac troponin T antibody (brown). (B) Muscle tissue with rat skeletal myoblasts. Muscle cells were immunostained with desmin antibody (brown). (C) Fat tissue with a hydrogel derived from adipose tissue extracellular matrix.31 Haematoxylin and eosin staining. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 3: Vascularity of a tissue constructs harvested at 4 weeks post-implantation. Representative images of lectin-stained sections. The blood vessels sprouting from the femoral artery (*) were labelled with lectin (brown). Scale bar = 200 µm (left) and 100 µm (right). Please click here to view a larger version of this figure.
Figure 4: Identification of transplanted cells in the tissue constructs. (A) DiI-label neonatal rat cardiomyocytes (red and white arrows) in a rat tissue construct harvested at 3 days post-implantation. (B) A representative human-specific Ku80 stained histology image of a tissue construct harvested from a rat tissue engineering chamber implanted with human induced pluripotent stem cells at 28 days post-implantation. Human nuclei were immuno-labelled brown and immunocompromized rat was used to prevent rejection of human cells. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Engineering of the microcirculation is currently being investigated essentially through two approaches. The first involves developing a highly interconnected vascular network within the construct in vitro so that when implanted, capillaries from the host vascular bed connect with those of the transplanted construct through a process called inosculation, thus ensuring the delivery of nutrients not only to the periphery but also to the core.21,32,33 This is called pre-vascularization. The second approach attempts to enhance the host's own vasculature directly in vivo, so that capillary sprouting occurs either before or concomitantly with implanted cell differentiation and tissue growth.17,34
The in vivo tissue engineering chamber protocol presented here exploits the latter concept by placing an artery and a vein, either as an arteriovenous loop or a flow-through pedicle configuration, inside a protected empty space, allowing significant sprouting and formation of new capillaries over time.3 Advantages of the chamber include (1) the absence of in vitro manipulations; (2) the generation of a completely autologous vascular network which will not be rejected by the host; and (3) the fact that it does not need an inosculation period which can take between 1 to 7 days rendering the tissue construct prone to ischemia.35,36
Nevertheless, it must be noted that it takes around 3-7 days for significant capillary sprouting to occur, a period during which the implanted tissue is also poorly supplied.3 Delayed cell implantation once the chamber is adequately vascularized has in fact shown improved survival.17 A further advantage includes the biocompatibility and non-immunogenicity of the chambers' material (i.e. polycarbonate and polyacrylic). In addition, the rigid non-collapsible chamber provides a protected space for the tissue and vasculature to grow without merging and integrating with the surrounding environment, which would otherwise hamper expansion and make harvest of the newly formed tissue difficult. Contrastingly, the fact that chambers are closed may limit tissue growth. This, however, has been addressed in the arteriovenous loop model, which now uses a perforated chamber that has been shown to grow tissue more effectively than the closed one.37
The tissue-engineering chamber protocols presented here are highly reproducible but it should be stressed that the chambers rarely fill to completion, usually to about 70% capacity. Consistent results are achieved provided that some critical steps and technical issues are taken into consideration. Pedicle patency is the ultimate aim when working with blood vessels, particularly if microsurgical anastomoses are performed. We have consistently found no tissue grows if the vascular pedicle thromboses early post-surgery. Factors affecting patency can be broadly grouped into four categories, namely surgical technical, blood flow, thrombosis and spasm.
First, a delicate surgical technique is the key to success. In this sense, it should be noted that these procedures, especially the one involving the creation of an arteriovenous loop require a certain level of surgical skill which can be readily acquired by practicing first in non-living models and subsequently in the rat's femoral vessels following the principles and techniques described here and elsewhere.38 Damage to the vessel wall, especially the intima, must be avoided at all times by proper handling of the vessels, which includes never grabbing the full thickness of the vessel wall, preventing excessive stretching, judicious use of the bipolar coagulator, and in the case of the arteriovenous loop, performance of meticulous and atraumatic microsurgical anastomoses. Although flushing with heparinized saline helps prevent clotting, it will never replace a fine surgical technique. Second, blood flow factors relate mainly to turbulence and stasis. Turbulent flow secondary to twists, bends or kinks of vessels promotes thrombus formation. Therefore a streamline unrestricted flow must be ensured in both the arteriovenous loop and the flow-through models. In this sense, the tethering effect of the epigastric branches in the arteriovenous loop model is essential to prevent bending; if for any reason these branches cannot be used, simple 10/0 nylon stitches from the vessel wall to the surrounding tissues should be carefully placed instead. Static blood at the anastomotic site during the arteriovenous loop procedure is also highly thrombogenic and must be prevented by flushing the vessel vigorously with heparinized saline prior to and throughout the anastomosis. Thirdly, pro-thrombotic factors such as contaminants from the operative field and most importantly the presence of pieces of adventitia inside the anastomosis are to be avoided. Preparing the vessel ends correctly prior to microsurgical suturing and keeping the field and anastomosis clean by flushing regularly are important aspects to consider when constructing the arteriovenous loop. Last but not least, there is the issue of vessel spasm. Even though the pathophysiology of vessel spasm has not been completely elucidated, it is likely to be due to both local and systemic factors. Local factors include vessel trauma, presence of blood in the operative field and tissue desiccation. Systemic factors, on the other hand, comprise low core temperature, hypotension and sympathetic response to pain.38
Thus, in both arteriovenous loop and flow-through models, proper handling and preparation of the animal together with a delicate surgical technique cannot be overemphasized. Strategies to resolve spasm include irrigation with warm saline or undiluted 1% xylocaine and having a rest period of 5-10 min. Vessel dilation and stripping of the adventitia can also help to relieve spasm due to its local sympathectomy effect.38 To circumvent the need to perform microsurgical anastomoses and the technical challenge it implies, the cuff technique can be employed as described elsewhere.39 This technique consists of inserting one of the vessel ends into a cuff, evert it and secure it with a circumferential 6/0 nylon suture. Next, the other vessel end is slid over the cuff and secured in a similar way.
The tissue-engineering chamber has opened a new window of possibilities in experimental research and is steadily advancing towards a potential clinical purpose. Until now, the models presented here have been used mainly for the generation of tissues of different lineages.4,7,8,17,22,,24,25,27-29,37,40 Nevertheless, they do have a number of other potential applications. For example, we have used the flow-through chamber as an effective and comparatively fast model for teratoma formation after implantation of human induced pluripotent stem cells.41,42 Thus, this approach may be used as a "quality control" tool to achieve tumor-free tissue engineering with pluripotent stem cells. Also, drug toxicology studies could be explored in human tissues grown inside the chamber. Likewise, generation of pathologic tissue could yield an interesting approach towards disease modeling and drug testing. Finally, the tissue-engineering chamber might also become a potential model to study growth of tissue and tracking of cell fate in vivo.
In conclusion, we have described a protocol involving the placement of a subcutaneous chamber in animals through two different approaches: using a microsurgical arteriovenous loop, or a flow-through pedicle configuration. The technique is highly reproducible and yields consistent results. Its use has been exploited mainly in the field of tissue engineering so far, however, there are other potential research fields for which these chambers might be of great application.
The authors have nothing to disclose.
This work was supported by grant funding from NHMRC and Stafford Fox Medical Foundation. The authors acknowledge the surgical assistance of Sue McKay, Liliana Pepe, Anna Deftereos and Amanda Rixon of the Experimental Medical and Surgical Unit, St. Vincent's Hospital, Melbourne. Support is also provided by the Victorian State Government's Department of Innovation, Industry and Regional Development's Operational Infrastructure Support Program.
1 15 Blade Scalpel | Braun | BB515 | |
1 Toothed Adson Forceps | Braun | BD527R | |
1 Needle Holder | Braun | BM201R | |
1 Bipolar Coagulator | Braun | US335 | |
1 Micro Needle Holder B-15-8.3 | S & T | 00763 | |
1 Micro Dilator Forceps D-5a.2 | S & T | 00125 | |
1 Micro Jeweler's Forceps JF-5 | S & T | 00108 | |
1 Micro Scissors – Straight SAS-11 | S & T | 00098 | |
1 Micro Scissors – Curved SDC-11 | S & T | 00090 | |
2 Single Clamps B-3 | S & T | 00400 | |
2 10/0 nylon suture | S & T | 03199 | |
1 6/0 nylon suture | Braun | G2095469 | |
2 4/0 Silk Sutures | Braun | C0760145 | |
Xilocaine 1% | Dealmed | 150733 | 10 mg/ml |
Heparin Sodium | Dealmed | 272301 | 5000 UI / ml |
Ringer Lactate | Baxter | JB2323 | 500 ml |
1 dome-shaped tissue engineering chamber | custom made | ||
1 flow-through chamber | custom made | ||
Lectin I, Griffonia Simplicifolia | Vector Laboratories | B-1105 | 1.67 μg/mL |
Troponin T antibody | Abcam | Ab8295 | 4 μg/mL |
Human-specific Ku80 antibody | Abcam | Ab80592 | 0.06 μg/mL |
Desmin antibody | Dako | M0760 | 2.55 μg/mL |
Cell Tracker CM-DiI dye | Thermo Fisher Scientific | C-7000 | 3 mg/106 cells |