Name: tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber
Uploaded: 2016-05-30T15:00:00
Description: Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

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 he...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+an+autologous+tissue+(matrix)+flap+by+combining+an+arteriovenous+shunt+loop+with+artificial+skin+in+rats:+preliminary+report.">Generation of an autologous tissue (matrix) flap by combining an arteriovenous shunt loop with artificial skin in rats: preliminary report.</a> B J Plast Surg. 53 (1), 51-57 (2000).</li><li>Cronin, K. J., 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=New+murine+model+of+spontaneous+autologous+tissue+engineering,+combining+an+arteriovenous+pedicle+with+matrix+materials.">New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials.</a> Plast Reconstr Surg. 113 (1), 260-269 (2004).</li><li>Forster, N. A., 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=A+prevascularized+tissue+engineering+chamber+supports+growth+and+function+of+islets+and+progenitor+cells+in+diabetic+mice.">A prevascularized tissue engineering chamber supports growth and function of islets and progenitor cells in diabetic mice.</a> Islets. 3 (5), 271-283 (2011).</li><li>Choi, Y. S., Matsuda, K., Dusting, G. J., Morrison, W. 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=Engineering+cardiac+tissue+in+vivo+from+human+adipose-derived+stem+cells.">Engineering cardiac tissue in vivo from human adipose-derived stem cells.</a> Biomaterials. 31 (8), 2236-2242 (2010).</li><li>Jeyaraj, R., G, N., Kirby, G., Rajadas, J., Mosahebi, A., Seifalian, A. 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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=Long-term+stability+of+adipose+tissue+generated+from+a+vascularized+pedicled+fat+flap+inside+a+chamber.">Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber.</a> Plast Reconstr Surg. 127 (6), 2283-2292 (2011).</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=Endothelial+cell+coculture+within+tissue-engineered+cardiomyocyte+sheets+enhances+neovascularization+and+improves+cardiac+function+of+ischemic+hearts.">Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts.</a> Circulation. 118, 145-152 (2008).</li><li>Ting, A. C., 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=The+adipogenic+potential+of+various+extracellular+matrices+under+the+influence+of+an+angiogenic+growth+factor+combination+in+a+mouse+tissue+engineering+chamber.">The adipogenic potential of various extracellular matrices under the influence of an angiogenic growth factor combination in a mouse tissue engineering chamber.</a> Acta Biomater. 10 (5), 1907-1918 (2014).</li><li>Zhan, W., 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=Self-synthesized+extracellular+matrix+contributes+to+mature+adipose+tissue+regeneration+in+a+tissue+engineering+chamber.">Self-synthesized extracellular matrix contributes to mature adipose tissue regeneration in a tissue engineering chamber.</a> Wound Repair Regen. 23 (3), 443-452 (2015).</li><li>Messina, A., Bortolotto, S. 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J., 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=Preparation+of+an+adipogenic+hydrogel+from+subcutaneous+adipose+tissue.">Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.</a> Acta Biomater. 9 (3), 5609-5620 (2013).</li><li>Dilley, R. J., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularisation+to+improve+translational+potential+of+tissue+engineering+systems+for+cardiac+repair.">Vascularisation to improve translational potential of tissue engineering systems for cardiac repair.</a> Int J Biochem Cell Biol. 56, 38-46 (2014).</li><li>Lesman, A., Koffler, J., Atlas, R., Blinder, Y. J., Kam, Z., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vessel-like+networks+within+multicellular+fibrin-based+constructs.">Engineering vessel-like networks within multicellular fibrin-based constructs.</a> Biomaterials. 32 (31), 7856-7869 (2011).</li><li>Hussey, A. J., 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=Seeding+of+pancreatic+islets+into+prevascularized+tissue+engineering+chambers.">Seeding of pancreatic islets into prevascularized tissue engineering chambers.</a> Tissue Eng Part A. 15 (12), 3823-3833 (2009).</li><li>Chen, X., Aledia, A. S., Popson, S. A., Him, L., Hughes, C. C., George, S. 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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 border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">1 15 Blade Scalpel</td>
			<td style="width:187px;">Braun</td>
			<td style="width:163px;">BB515</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Toothed Adson Forceps</td>
			<td>Braun</td>
			<td>BD527R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Needle Holder</td>
			<td>Braun</td>
			<td>BM201R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Bipolar Coagulator&#160;</td>
			<td>Braun</td>
			<td>US335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Needle Holder B-15-8.3</td>
			<td>S &#38; T</td>
			<td>00763</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Dilator Forceps D-5a.2</td>
			<td>S &#38; T</td>
			<td>00125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Jeweler&#39;s Forceps JF-5</td>
			<td>S &#38; T</td>
			<td>00108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Straight SAS-11</td>
			<td>S &#38; T</td>
			<td>00098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Curved SDC-11</td>
			<td>S &#38; T</td>
			<td>00090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 Single Clamps B-3</td>
			<td>S &#38; T</td>
			<td>00400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 10/0 nylon suture</td>
			<td>S &#38; T</td>
			<td>03199</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 6/0 nylon suture</td>
			<td>Braun</td>
			<td>G2095469</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 4/0 Silk Sutures</td>
			<td>Braun</td>
			<td>C0760145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xilocaine 1%</td>
			<td>Dealmed</td>
			<td>150733</td>
			<td>10 mg/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin Sodium</td>
			<td>Dealmed</td>
			<td>272301</td>
			<td>5000 UI / ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ringer Lactate</td>
			<td>Baxter</td>
			<td>JB2323</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 dome-shaped tissue engineering chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 flow-through chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin I, Griffonia Simplicifolia&#160;</td>
			<td>Vector Laboratories</td>
			<td>B-1105</td>
			<td>1.67 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Troponin T antibody</td>
			<td>Abcam</td>
			<td>Ab8295</td>
			<td>4 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human-specific Ku80 antibody</td>
			<td>Abcam</td>
			<td>Ab80592</td>
			<td style="width:176px;">0.06 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Desmin antibody</td>
			<td>Dako</td>
			<td>M0760</td>
			<td style="width:176px;">2.55 &#956;g/mL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Tracker CM-DiI dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C-7000</td>
			<td>3 mg/106 cells</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 ...

The overall goal of this procedure is to create a space where tissue can grow and expand concomitantly with the development of a robust blood supply, ultimately leading to the generation of a vascularized, large, three-dimensional block of tissue. This method can help answer key questions in the engineering of vascularized tissue for reconstructive purposes as well as for organ replacement and repair. The main advantage of this technique is that the newly generated tissue is intrinsically supplied by its own vascular network without having to rely on diffusion from the environment for survival.Demonstrating the procedure will be Sue McKay and Liliana Pepe. Anesthetize a rat weighing at least 250 grams, and assess the depth of anesthesia using a toe pinch. If the rat is unresponsive, maintain its sedation using 2%isoflurane throughout the procedure.Next, apply epsomic ointment to the eyes. Then, with the rat supine, use electric razors to shave the groin and remove the hair with moistened gauze. Then, clean the surgical sites with a chlorhexidine solution containing 70%ethanol.Finally, drape the animal with sterile towels and proceed with the surgery. Using a number 15 blade, make a four centimeter long skin incision into the left groin parallel to the inguinal ligament. Next, cut through the exposed inguinal fat pad circumferentially with scissors, leaving it attached to its vascular pedicle, based on the epigastric vessels.Now, with micro-scissors, free the filmy connective tissue adhesions between the abdominal wall and underlying femoral vessels. Then, place a retractor on the abdominal wall and pull it medially to expose the inguinal ligament and the whole length of the femoral vessels. Next, using micro-forceps and curved scissors, dissect the epigastric vein and isolate it from the surrounding fat.This vein will function as a tether. Proceed by opening the perivascular sheath containing the femoral vessels and nerve. Open it all the way from the inguinal ligament to its bifurcation distal to the epigastric branch.At this point, and throughout the whole procedure, gentle handling of the femoral vessels is critical. Their damage can result in thrombosis and failure of the whole experiment. Then, using micro-forceps, pick up the femoral vein by its adventitia and gently separate it from the surrounding tissues and the accompanying artery.During this step, do not grasp the whole thickness of the vein wall, which could cause trauma to the intima, thus making the vein prone to thrombosis. Also during this step, ligate the side branches with 10-0 nylon suture, or coagulate them with a bipolar coagulator. With the femoral vein completely free, ligate it proximally and distally with 4-0 silk sutures to obtain a vein graft that is at least 10 millimeters long, which includes about half a centimeter length of the epigastric branch.The branch works as a guy rope tether to hold the loop open in the chamber. Next, trim the adventitia from the graft's ends. If necessary, this step may be postponed to before the micro-surgical anastomoses.Finish preparing the vein graft by flushing it with heparinized saline solution and leaving it to rest loaded with heparinized saline solution. Then, close the wound using continuous running 4-0 silk suture and two or three additional simple interrupted stitches. To begin, expose the inguinal ligament and the whole length of the femoral vessel of the contralateral limb as previously demonstrated.Then, using micro-forceps, dissect and isolate both the epigastric artery and vein from the surrounding fat pad. Be gentle when pulling the tissue away from the vessels. Continue by picking up the femoral artery by its adventitia and freeing it from the surrounding tissues.In this process, ligate or coagulate the artery side branches. Then, place a clamp proximally on both the femoral artery and vein. Now, using 4-0 silk suture, ligate the femoral artery and vein distal to the emergence of the epigastric vessels.Now place a sterile plastic contrast background under the vessels and use a sharp, straight micro-scissor to cut each vessel transversely. Once cut, flush the vessels out vigorously with generous amounts of heparinized saline until all the blood has been removed from the lumen. Now bring the vein graft into the operative field.Position the vein graft so that the proximal end of the vein graft is ready to be anastomosed to the recipient femoral vein, and the distal end to the recipient femoral artery. This will allow the blood to flow from the arterial to the venous side without resistance from the valves inside the vein graft. Make sure the femoral vessels and the vein graft rest in their natural position without any twists.Using 10-0 nylon suture, perform surgical anastomosis at both sides. A good and atraumatic micro-surgical anastomosis is key. Damage to the vessel intima, dessication of the vessels, or incorrectly placed stitches make the anastomotic site prone to thrombus formation.Then, 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 or a sponge on top and gently compressing for five to 10 minutes. Larger pulsating leaks that rapidly flood the entire field will need additional stitches.Next, check the patency of the arteriovenous loop. Gentle occlusion of the femoral artery should make it shrink. Conversely, occlusion of the femoral vein should engorge the loop.Now place the base of the tissue engineering chamber under the arteriovenous loop. Make sure that the loop rests in its natural position without any twists or kinks. Then, with 6-0 nylon sutures, secure the base of the chamber to the inguinal ligament and the underlying muscle fascia.Next, place the lid over the base of the chamber. Pinch the epigastric branches under the lid to hold the loop in position. Then, close the wound using continuous running 4-0 silk suture, plus two or three additional simple interrupted stitches.Finally, allow the animal to recover from anesthesia in an individual cage, and administer a single dose of carprofen subcutaneously as an analgesic. Prepare the animal for surgery as before. Two chambers can be implanted into a single rat, one in each groin.Then, isolate the femoral vessel as previously described. With both the artery and vein completely freed of surrounding tissues and their branches ligated, bring the chamber into the operative field. Then, place each of the intact femoral vessels into the corresponding slits of the chamber base.Make sure that neither is twisted or kinked. Next, close the chamber by attaching the lid to the base. Then close the wound and allow the animal to recover as previously described.The micro-surgical creation of tissue engineering chambers was performed as described. Various tissue types were successfully engineered using the in vivo vascularized chamber, which include cardiac tissue with neonatal rat cardiomyocytes, muscle tissue with rat skeletal myoblasts, and adipose tissue with hydrogel derived from adipose tissue extracellular matrix. The stereology system allows for unbiased quantification of specific tissue volume.For example, lectin-stained endothelial cells in transverse sections were used to estimate vascular volumes. Following in vivo implantation of cells labeled with fluorescent dyes, their fate can be tracked. For example, neonatal rat cardiomyocytes labeled with dye i were detected in the tissue constructs three days post-implantation.Alternatively, species-specific antibodies can be used to identify implanted cells in xenotransplanation studies. For example, human-induced pluripotent stem cells implanted in immuno-compromised rats could be identified in the tissue harvested from the chamber. Once mastered, the flow-through chamber and the arteriovenous loop can be implanted in approximately 20 and 90 minutes, respectively.While attempting this procedure, it's important to maintain sterility at all times and take the necessary measures should contamination occur. Don't forget that gentle handling of the blood vessels and atraumatic micro-surgical anastomosis are key to ensure success. In this sense, we do recommend basic micro-surgical training before attempting this procedure, particularly for the construction of the arteriovenous loop.

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

Research

JoVE Journal

Bioengineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

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 ...

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. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

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

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

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

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...