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&#39;s Hospital, Melbourne. Support is also provided by the Victorian State Government&#39;s Department of Innovation, Industry and Regional Development&#39;s Operational Infrastructure Support Program.

The protocols described here have been approved by the Animal Ethics Committee of St. Vincent&#39;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.
<ol>
	<li>Preparation of the Animal for Surgery
	<ol>
		<li>Use rats weighing at least 250 g for their large size of vessels for creation of the arteriovenous loop.</li>
		<li>Anaesthetize the animal with 4% isoflurane inhalation. Corroborate adequate depth of anesthesia by assessing unresponsiveness to toe-pinch. After anesthesia, keep the animal adequately anesthetized&#160;throughout the procedure with 2% isoflurane.</li>
		<li>Place the animal in a supine position on a warming pad and apply sterile lubricant to the eyes to prevent desiccation during surgery.</li>
		<li>Using an electric razor, shave both groins and remove hair with a piece of moist gauze.</li>
		<li>Prep the surgical sites with chlorhexidine/70% ethanol solution and drape the animal with sterile towels.&#160;Administer a single dose of Carprofen (5 mg/kg, subcutaneously) as analgesic.</li>
	</ol>
	</li>
	<li>Harvest of Femoral Vein Graft
	<ol>
		<li>Using a #15 blade, make a 4 cm long skin incision on the left groin parallel to the inguinal ligament. This exposes the inguinal fat pad.</li>
		<li>Cut through the fat pad circumferentially with scissors leaving it attached to its vascular pedicle based on the epigastric vessels.</li>
		<li>Using micro scissors, free the filmy connective tissue adhesions between the abdominal wall and underlying femoral vessels.</li>
		<li>Place a retractor on the abdominal wall and pull medially. This exposes the inguinal ligament and the whole length of the femoral vessels.</li>
		<li>Using micro forceps and curved scissors dissect the epigastric vein and isolate it from its surrounding fat by gently pulling and cutting. This vein acts as a tether when constructing the loop.</li>
		<li>Using micro forceps and curved micro scissors open the perivascular sheath containing the femoral vessels and nerve all the way from the inguinal ligament to its bifurcation distal to the epigastric branch.</li>
		<li>Using micro forceps, pick up the femoral vein by its adventitia and gently separate it from the surrounding tissues and accompanying artery. Do this with micro forceps and curved round-pointed micro scissors by pulling the tissue apart and cutting through it. 
		NOTE: Never grab the whole thickness of the vein wall as this might cause trauma to the intima making it prone to thrombosis.</li>
		<li>Ligate side branches found during the dissection with 10/0 nylon suture or coagulate them with a bipolar coagulator.</li>
		<li>With the femoral vein completely free, ligate its proximal and distal ends with 4/0 silk sutures. Make sure to obtain a vein graft of at least 10 mm length and include approximately 0.5 cm length of the epigastric branch to be used as a guy rope tether to hold the loop open in the chamber.</li>
		<li>Using micro forceps and straight micro scissors, trim the adventitia from the graft&#39;s ends by gently pulling and cutting. This can also be done later, before microsurgical anastomoses.</li>
		<li>Flush the vein graft with heparinized saline solution (10 U/ml of heparin) and leave it to rest in the solution. Close the wound using continuous running 4/0 silk suture plus two or three additional simple interrupted stitches.</li>
	</ol>
	</li>
	<li>Creation of Arteriovenous Loop and Implantation of Chamber
	<ol>
		<li>Repeat steps 1.2.1 to 1.2.4 in the exact same way on the contralateral limb.</li>
		<li>Using micro forceps, dissect and isolate both the epigastric artery and vein form the surrounding fat pad. Do this by gently pulling the tissue away from the vessels</li>
		<li>Using micro forceps, pick up the femoral artery by its adventitia and free it from the surrounding tissues. Do this with micro forceps and curved round-pointed micro scissors by pulling the tissue apart and cutting through it. Ligate or coagulate its side branches.</li>
		<li>Ligate the femoral artery and vein distal to the emergence of the epigastric vessels using 4/0 silk suture.</li>
		<li>Place a single clamp proximally on each of the femoral artery and vein. Using a sharp straight micro scissor, make a clean transverse cut in each vessel distal to the emergence of the epigastric branches. Place a sterile plastic contrast background under the vessels.</li>
		<li>Flush the vessels vigorously with generous amounts of heparinized saline until all the blood is removed from the lumen.</li>
		<li>Bring the vein graft into the operative field and remove any redundant adventitia from the vessels&#39; ends as per step 1.2.10, if needed.</li>
		<li>Perform both microsurgical anastomoses with 10/0 nylon suture. Anastomose the proximal end of the vein graft to the femoral vein and the distal end to the femoral artery. This will allow the blood to flow from the arterial to the venous side without resistance from valves inside the vein graft. 
		NOTE: Make sure the femoral vessels and the vein graft rest in their natural position without any twists.</li>
		<li>Check for leaks at both anastomotic sites. Resolve small leaks, which look like non-pulsating blood coming out of the anastomotic site, by placing a small piece of fat on top and gently compressing for 5-10 min. Larger pulsating leaks that rapidly flood the entire field will need additional stitches.</li>
		<li>Check patency of the arteriovenous loop. Gentle occlusion of the femoral artery should make it shrink while the same in the femoral vein should engorge it.</li>
		<li>Place the base of the tissue engineering chamber under the arteriovenous loop with the latter resting in its natural position without twists or kinks.</li>
		<li>Secure the base of the chamber to the inguinal ligament and underlying muscle fascia with 6/0 nylon sutures.</li>
		<li>Place the lid over the base so that the femoral vessels enter the chamber through a notch (window in the side of the chamber). When closing the lid, make sure it catches the epigastric branches, between the chamber base and lid, which act as tethers to hold the arteriovenous loop into position.</li>
		<li>Close the wound using continuous running 4/0 silk suture plus two or three additional simple interrupted stitches.</li>
		<li>Allow the animal to recover from anesthesia on a warming pad.&#160;</li>
		<li>Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Likewise, do not return an animal that has undergone surgery to the company of other animals until fully recovered. 24 hr&#160;later, administer another single dose of Carprofen (5 mg/kg, subcutaneously) as analgesic.</li>
		<li>Treat the wound with topical antibiotic ointment for 5 days. If the wound is opened, anesthetize the animal as in step 1.1.2 through 1.1.5 and close the wound as in 1.3.14. Monitor the health of animal daily. Euthanize the animal using a lethal dose of intraperitoneal lethabarb injection (163 mg/kg in 0.25 ml by 23 G needle) if the animal shows more than one moderate signs of inacitivity, poor appetite, weight loss and loss of color.</li>
	</ol>
	</li>
</ol>
2. Flow-through Pedicle Chamber (Two Chambers per Animal)
<ol>
	<li>Preparation of the Animal for Surgery
	<ol>
		<li>Repeat steps 1.1.1 through 1.1.4. Two chambers can be implanted into both groin regions of a single rat.</li>
	</ol>
	</li>
	<li>Isolation of Femoral Vessels and Insertion of the Chamber
	<ol>
		<li>Repeat steps 1.2.1 through 1.2.8.</li>
		<li>With both artery and vein completely freed of surrounding tissues and their branches ligated, bring the chamber into the operative field.</li>
		<li>Place each of the intact femoral vessels on the corresponding slit of the chamber base making sure there are no twists or kinks.</li>
		<li>Close the chamber by attaching the lid to the base. Close the wound using continuous running 4/0 silk suture plus two or three additional simple interrupted stitches and allow the animal to recover as previously described.</li>
	</ol>
	</li>
</ol>
3. Harvest of Chambers and Tissue Processing
<ol>
	<li>Once the experiment&#39;s time points (4-6 weeks post-implantation) are reached, anesthetize the animal as in step 1.1.2 and repeat steps 1.1.3 through 1.1.5.</li>
	<li>Open the wound using a #15 blade and cut through the tissues with scissors until the chamber is completely exposed.</li>
	<li>Expose the femoral vessels proximal to the construct and test for vascular patency: gently occlude the vessel with two microforceps, then milk the blood in a distal direction and finally release the proximal forceps. If the vessel fills with blood again, this confirms patency. Ligate the femoral vessels proximally in the case of the arteriovenous loop and both proximally and distally in the case of the flow-through configuration, and remove the chambers with the containing tissue en bloc.</li>
	<li>At the end of the experiment, euthanize the animal using a lethal dose of intraperitoneal lethobarb injection (163 mg/kg in 0.25 ml by 23 G needle).</li>
	<li>Fix tissues in 4% paraformaldehyde at room temperature for 24 hr. Divide tissues into multiple transverse sections (1-2 mm thick) and embed in paraffin wax or optimal cutting temperature compound for paraffin sections (5 &#181;m) or frozen sections (10 &#181;m), respectively.3,4,8,17,22,24</li>
	<li>Perform routine histological staining such as hematoxylin and eosin to examine the general morphology of tissues. Perform immunohistochemical staining with specific antibody to identify cell type of interest,3,4,8,17,22,24,29 for example cardiac troponin T immunostaining for cardiomyocytes.</li>
</ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+large+volume+adipose+tissue+generation+from+a+vascularized+pedicled+fat+flap+inside+a+chamber+space.">Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space.</a> Tissue Eng. 13 (4), 673-681 (2007).</li><li>Wei, F. C., Lin Tay, S. K., Neligan, P. C., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+techniques+of+microvascular+surgery.">Principles and techniques of microvascular surgery.</a> Plastic Surgery. Volume 1. , 587-620 (2013).</li><li>Sekine, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fabrication+of+functional+three-dimensional+tissues+with+perfusable+blood+vessels.">In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels.</a> Nat.Comm. 4 (1399), 1-10 (2013).</li><li>Lim, S. Y., Sivakumaran, P., Crombie, D. E., Dusting, G. J., P&#233;bay, A., Dilley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichostatin+A+enhances+differentiation+of+human+induced+pluripotent+stem+cells+to+cardiogenic+cells+for+cardiac+tissue+engineering.">Trichostatin A enhances differentiation of human induced pluripotent stem cells to cardiogenic cells for cardiac tissue engineering.</a> Stem Cells Transl Med. 2 (9), 715-725 (2013).</li><li>Lim, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+tissue+engineering+chamber+supports+human+induced+pluripotent+stem+cell+survival+and+rapid+differentiation.">In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.</a> Biochem Biophys Res Commun. 422 (1), 75-79 (2012).</li><li>Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+integration-free+human+induced+pluripotent+stem+cells+from+keratinocytes+by+simple+transfection+of+episomal+vectors.">Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.</a> Stem Cells Transl Med. 3 (7), 787-791 (2014).</li></ol>

The authors declare no competing interests.

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 approac...

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 (&#60;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.

<table><tbody><tr><td>1 15 Blade Scalpel</td><td>Braun</td><td>BB515</td><td></td></tr><tr><td>1 Toothed Adson Forceps</td><td>Braun</td><td>BD527R</td><td></td></tr><tr><td>1 Needle Holder</td><td>Braun</td><td>BM201R</td><td></td></tr><tr><td>1 Bipolar Coagulator&amp;nbsp;</td><td>Braun</td><td>US335</td><td></td></tr><tr><td>1 Micro Needle Holder B-15-8.3</td><td>S &amp;amp; T</td><td>00763</td><td></td></tr><tr><td>1 Micro Dilator Forceps D-5a.2</td><td>S &amp;amp; T</td><td>00125</td><td></td></tr><tr><td>1 Micro Jeweler&#039;s Forceps JF-5</td><td>S &amp;amp; T</td><td>00108</td><td></td></tr><tr><td>1 Micro Scissors - Straight SAS-11</td><td>S &amp;amp; T</td><td>00098</td><td></td></tr><tr><td>1 Micro Scissors - Curved SDC-11</td><td>S &amp;amp; T</td><td>00090</td><td></td></tr><tr><td>2 Single Clamps B-3</td><td>S &amp;amp; T</td><td>00400</td><td></td></tr><tr><td>2 10/0 nylon suture</td><td>S &amp;amp; T</td><td>03199</td><td></td></tr><tr><td>1 6/0 nylon suture</td><td>Braun</td><td>G2095469</td><td></td></tr><tr><td>2 4/0 Silk Sutures</td><td>Braun</td><td>C0760145</td><td></td></tr><tr><td>Xilocaine 1%</td><td>Dealmed</td><td>150733</td><td>10 mg/ml</td></tr><tr><td>Heparin Sodium</td><td>Dealmed</td><td>272301</td><td>5,000 UI/ml</td></tr><tr><td>Ringer Lactate</td><td>Baxter</td><td>JB2323</td><td>500 ml</td></tr><tr><td>1 dome-shaped tissue engineering chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>1 flow-through chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>Lectin I, Griffonia Simplicifolia&amp;nbsp;</td><td>Vector Laboratories</td><td>B-1105</td><td>1.67 &amp;mu;g/ml</td></tr><tr><td>Troponin T antibody</td><td>Abcam</td><td>Ab8295</td><td>4 &amp;mu;g/ml</td></tr><tr><td>Human-specific Ku80 antibody</td><td>Abcam</td><td>Ab80592</td><td>0.06 &amp;mu;g/ml</td></tr><tr><td>Desmin antibody</td><td>Dako</td><td>M0760</td><td>2.55 &amp;mu;g/ml</td></tr><tr><td>Cell Tracker CM-DiI dye</td><td>Thermo Fisher Scientific</td><td>C-7000</td><td>3 mg/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Lethabarb (sodium pentobarbitone)</td><td><a href="www.virbac.com.au" target="_blank"><img src="/img/new_window.png" /> Virbac Animal Health</a></td><td>LETHA450</td><td>325 mg/ml</td></tr><tr><td>Heat pad flexible</td><td><a href="http://www.redzoneheating.com.au" target="_blank"><img src="/img/new_window.png" /> Redzone Heating</a></td><td>RZ/Medium</td><td></td></tr></tbody></table>

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.

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...

Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

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.

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 ...

tissue-engineering-intrinsic-vascularization-an-vivo-tissue

Research

JoVE Journal

Bioengineering

8.8K Views.  St Vincent's Institute of Medical Research. 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. 

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

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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 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.

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

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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 ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Biology

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

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

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Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures