The authors would like to acknowledge the staff at the Department of Experimental Medicine (AEM), University of Copenhagen, for assistance with planning and carrying out animal surgeries and husbandry, and ELLA-CS, s.r.o, Hradec Kralove, Czech Republic, for providing customized biodegradable stents used in the study. Financial support was provided by the Swedish Society of Medical Research, Promobilia Foundation, Rydbeck Foundation, Samariten Foundation, The Foundation for Pediatric Health Care, Foundation Frimurare Barnhuset in Stockholm, and the Novo Nordisk Foundation (NNFSA170030576).

This experiment was carried out in an AAALAC accredited experimental facility in accordance with the European legislation on laboratory use of animal subjects and after ethical permission granted by the Danish Ministry of Food and Agriculture (Ref. no. 2022-15-0201-01206).
1. Surgical procedure
<ol>
	<li>Animal preparation
	<ol>
		<li>Fast a female full-grown G&#246;ttingen minipig for at least 12 h preoperatively.</li>
		<li>Prepare the surgical table with all sterile utensils as described below.</li>
		<li>For full-grown standard-size minipigs, sedate the animal by intramuscular injection with 1.0-1.4 mL/10 kg with a solution of 125 mg of zolazepam and 125 mg of tiletamine suspended in 1.25 mL of ketamine (100 mg/mL), 6.25 mL of xylazine (20 mg/mL), 1.25 mL of methadone (10 mg/mL) and 2 mL of butorphanol (10 mg/mL) (later on referred as sedation mixture).</li>
		<li>Perform visual-guided endotracheal intubation and add capnography as an anesthesia monitoring parameter and confirm proper tube placement. Confirm anesthesia by vital signs and eye and interdigital reflex testing. Apply ophthalmic ointment bilaterally.</li>
		<li>Install bilateral ear vein catheters and sustain anesthesia with propofol (10-15 mg/kg/h) and fentanyl (5-15 mg/kg/h).</li>
		<li>Insert an 8 Fr urinary catheter and fill the bladder with 250 mL of physiologically temperate isotonic saline using an appropriately sized luer lock syringe.</li>
		<li>Place the pig in the supine position, then raze and scrub the abdomen. After two further rounds of skin cleaning with 70% ethanol and chlorhexidine scrub in alternating rounds.&#160;Frame the surgical field with sterile draping.</li>
	</ol>
	</li>
	<li>Tissue harvesting and surgical scaffold implantation&#160;
	<ol>
		<li>Perform a standard lower midline laparotomy with scalpel and cautery, dividing the skin, muscle, and peritoneum, and pull the intraperitoneal urinary bladder to the incision.</li>
		<li>Perform prophylactic hemostasis with electrocautery on the anterior bladder wall and excise a 2 cm2 full-wall segment, leaving a proximal opening of 1 cm2 while closing the remaining bladder wall with a fast-resorbable monofilament&#160;running suture.</li>
		<li>Carefully dissect the mucosal layer of the resected specimen and mince a 2 cm2 mucosal specimen into 1 mm2 micrografts for scaffold embedding (described below in section 2).</li>
		<li>After completing the scaffold, anastomose the tubular construct to the remaining opening on the anterior bladder wall with a slow-resorbable monofilament running suture.</li>
		<li>Use a peritoneal flap from the pubovesical ligament to patch the tubular scaffold and place an intraluminal 14 Fr antegrade colonic enema (ACE) stopper into the tubular scaffold.</li>
		<li>Ligate the distal end of the conduit with a slow-resorbable 4-0 monofilament suture to prevent urine from leaking out, and inject a total of 250 mL of sterile saline with syringes via the bladder catheter to confirm anastomotic patency.</li>
		<li>Bluntly dissect a trans-fascial channel laterally to the midline, 2-3 cm caudally to the caudal mammary gland on the right side, and place the conduit in a subcutaneous pocket. Fixate the distal conduit with two transcutaneous non-resorbable monofilament sutures to mark the location at skin level.</li>
		<li>Close the anterior muscle fascia of the abdominal muscle with a slow resorbable monofilament running suture, adapt the subcutis with a fast-resorbable braided running suture, and close the skin with a non-resorbable monofilament running suture.</li>
		<li>After discontinuing anesthesia, extubate the animal and observe it in the stables until it is fully ambulant and safely able to drink and eat.</li>
	</ol>
	</li>
</ol>
2. Scaffold construction
<ol>
	<li>Preparation of the composite scaffold
	<ol>
		<li>Prior to surgery (maximum 2 h), prepare a liquid solution of rat-tail collagen type I as previously described17. In brief, add 4:1 of 10x minimum essential medium (MEM) to the collagen solution and approximate the pH to 7.4 with 1 M NaOH, and finally add 1x MEM, aiming for a final collagen concentration of 1.64 mg/mL. Store the solution in a sterile vial on ice until further use.</li>
		<li>After surgical tissue resection and mincing, manually place the mucosal particles (i.e., micrografts) onto a 2 cm x 6 cm fitted biodegradable mesh with a 1:6 expansion rate (e.g., a 2 cm2 mucosal tissue is expanded to a 12 cm2 mesh) using forceps.</li>
		<li>Prepare a sterile rectangular steel mold measuring 1 cm x 3 cm x 6 cm (height x width x length) on top of a sterile steel plate and place the mesh into the steel mold with the micrografts facing upwards. Gently pour 20 mL of the collagen solution into the mold, making sure not to flush the micrografts off the mesh. Transfer the entire construct to a 38 &#176;C sterile heating chamber and leave to solidify for five minutes.</li>
		<li>After sufficient solidification, slide the hydrogel onto a nylon mesh resting on a perforated steel plate and gently remove the mold.</li>
		<li>Expel water from the hydrogel by placing a nylon mesh and then a steel plate on top of the gel, and then passively compress with a 120 g weight (in this case equivalent to the steel mold used for embedding) placed on top of the steel plate for 5 min.</li>
		<li>After compression, roll the flattened scaffold around a biodegradable stent, micrografts facing the stent, measuring 5 cm x 0.6 cm (length x inner diameter), and suture the scaffold in place longitudinally with a slow-resorbable monofilament running suture. The completed conduit is now ready for surgical implantation.</li>
	</ol>
	</li>
</ol>
3. Postoperative management
<ol>
	<li>Analgesia and antibiotic prophylaxis
	<ol>
		<li>Administer buprenorphine (0.05-0.1 mg/kg/8 h intravenously) for the first 3 days, meloxicam (0.4 mg/kg/day intramuscular or orally) for the first 4 days, and trimethoprim (2.7 mg/kg/day intramuscular or 4.2 mg/kg/day orally) and sulfadoxin (13.3 mg/kg/day intramuscular or 20.8 mg/kg/day orally) for the first 5 days. Administer the intramuscular injections postoperatively while the animal is still anesthetized.</li>
		<li>Single-house the animals to avoid nibbling of external vein catheters and suture material. Provide visual contact with neighboring minipigs through plexiglass windows and the possibility of snout contact between pens. Provide daily fresh straw and hay, as well as toys and water supply ad libitum and feed twice daily.</li>
		<li>Monitor the animals daily for natural behavior, eating habits, urine- and stool production, and assess body weight weekly.</li>
		<li>At the end of the observational period (6 weeks), sedate the animals with 1-1.4 mL/10 kg intramuscular injection of sedation mixture and terminate the animal with a lethal pentobarbital injection (100 mg/kg intravenously).</li>
	</ol>
	</li>
</ol>
4. Postmortem assessments
<ol>
	<li>Gross anatomy
	<ol>
		<li>After termination, dissect the distal conduit at skin level and remove the ACE stopper. Close the urethra with a plastic clamp and inject 250 mL of a 1:20 contrast solution of iohexol in isotonic saline via the distal conduit opening using a catheter.</li>
		<li>Assess the animal with a 64-slice computed tomography scanner. Visualize images using multiplanar reconstruction and analyze all images using medical image processing software.</li>
		<li>Perform an endoscopic examination of the bladder and conduit lumina with a 16.2 Fr flexible cystoscope via the native urethra.</li>
		<li>Resect the conduit en bloc while carefully evaluating any gross anatomical findings. Additionally, resect full-wall bladder biopsies with a 2 cm margin to the conduit anastomosis and process in a similar fashion for reference values.</li>
	</ol>
	</li>
	<li>Histological processing
	<ol>
		<li>Fix the excised specimen in 10% formalin for 24 h.</li>
		<li>Divide the conduit orthogonally with a scalpel into equal-sized separate sections of proximal, medial, and distal conduit segments. Dehydrate the specimens with increasing ethanol concentrations and embed them in paraffin before microtome sectioning.</li>
		<li>Stain 5 &#181;m sections with hematoxylin and eosin (H&#38;E) and pancytokeratin CK-AE and scan with a digital histology slide scanner.</li>
	</ol>
	</li>
</ol>

Accessing Collagen Scaffold for Single-Stage Urinary Diversion Surgery in Pigs

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+gastric+augmentation+on+bladder+function.">The effect of gastric augmentation on bladder function.</a> J Urol. 149 (5), 1099-1102 (1993).</li><li>Husmann, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+following+augmentation+cystoplasty:+A+transitional+urologist's+viewpoint.">Mortality following augmentation cystoplasty: A transitional urologist's viewpoint.</a> J Pediatr Urol. 13 (4), 358-364 (2017).</li><li>Mitrofanoff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-appendicular+continent+cystostomy+in+the+management+of+the+neurogenic+bladder.">Trans-appendicular continent cystostomy in the management of the neurogenic bladder.</a> Chir Pediatr. 21 (4), 297-305 (1980).</li><li>Leslie, B., Lorenzo, A. J., Moore, K., Farhat, W. A., B&#228;gli, D. J., Pippi Salle, J. L. <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+followup+and+time+to+event+outcome+analysis+of+continent+catheterizable+channels.">Long-term followup and time to event outcome analysis of continent catheterizable channels.</a> J Urol. 185 (6), 2298-2302 (2011).</li><li>Horst, M., Eberli, D., Gobet, R., Salemi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+in+pediatric+bladder+reconstruction-The+road+to+success.">Tissue engineering in pediatric bladder reconstruction-The road to success.</a> Front Pediatr. 7, 91 (2019).</li><li>Ajalloueian, F., Lemon, G., Hilborn, J., Chronakis, I. S., Fossum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+biomechanics+and+the+use+of+scaffolds+for+regenerative+medicine+in+the+urinary+bladder.">Bladder biomechanics and the use of scaffolds for regenerative medicine in the urinary bladder.</a> Nat Rev Uro. 15 (3), 155-174 (2018).</li><li>Dorin, R. P., Pohl, H. G., De Filippo, R. E., Yoo, J. J., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubularized+urethral+replacement+with+unseeded+matrices:+what+is+the+maximum+distance+for+normal+tissue+regeneration.">Tubularized urethral replacement with unseeded matrices: what is the maximum distance for normal tissue regeneration.</a> World J Uro. 26 (4), 323-326 (2008).</li><li>El Kassaby, A. W., AbouShwareb, T., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomized+comparative+study+between+buccal+mucosal+and+acellular+bladder+matrix+grafts+in+complex+anterior+urethral+strictures.">Randomized comparative study between buccal mucosal and acellular bladder matrix grafts in complex anterior urethral strictures.</a> J Urol. 179 (4), 1432-1436 (2008).</li><li>Casarin, M., 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=Porcine+small+intestinal+submucosa+(SIS)+as+a+suitable+scaffold+for+the+creation+of+a+tissue-engineered+urinary+conduit:+Decellularization,+biomechanical+and+biocompatibility+characterization+using+new+approaches.">Porcine small intestinal submucosa (SIS) as a suitable scaffold for the creation of a tissue-engineered urinary conduit: Decellularization, biomechanical and biocompatibility characterization using new approaches.</a> Int J Mol Sci. 23 (5), 2826 (2022).</li><li>Casarin, M., 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+novel+hybrid+membrane+for+urinary+conduit+substitutes+based+on+small+intestinal+submucosa+coupled+with+two+synthetic+polymers.">A novel hybrid membrane for urinary conduit substitutes based on small intestinal submucosa coupled with two synthetic polymers.</a> J Funct Biomater. 13 (4), 222 (2022).</li><li>Drewa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+artificial+conduit+for+urinary+diversion+in+rats:+a+preliminary+study.">The artificial conduit for urinary diversion in rats: a preliminary study.</a> Transplant Proc. 39 (5), 1647-1651 (2007).</li><li>Liao, 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=Tissue-engineered+tubular+graft+for+urinary+diversion+after+radical+cystectomy+in+rabbits.">Tissue-engineered tubular graft for urinary diversion after radical cystectomy in rabbits.</a> J Surg Res. 182 (2), 185-191 (2013).</li><li>Reinfeldt Engberg, G., Lundberg, J., Chamorro, C. I., Nordenskj&#246;ld, A., Fossum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+autologous+minced+bladder+mucosa+for+a+one-step+reconstruction+of+a+tissue+engineered+bladder+conduit.">Transplantation of autologous minced bladder mucosa for a one-step reconstruction of a tissue engineered bladder conduit.</a> Biomed Res Int. 2013, 212734 (2013).</li><li>Ajalloueian, F., Nikogeorgos, N., Ajalloueian, A., Fossum, M., Lee, S., Chronakis, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compressed+collagen+constructs+with+optimized+mechanical+properties+and+cell+interactions+for+tissue+engineering+applications.">Compressed collagen constructs with optimized mechanical properties and cell interactions for tissue engineering applications.</a> Int J Biol Macromol. 108, 158-166 (2018).</li><li>Chamorro, C. I., Zeiai, S., Engberg, G. R., Fossum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minced+tissue+in+compressed+collagen:+A+cell-containing+biotransplant+for+single-staged+reconstructive+repair.">Minced tissue in compressed collagen: A cell-containing biotransplant for single-staged reconstructive repair.</a> J Vis Exp. (108), e53061 (2016).</li><li>Juul, N., 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=Insights+into+cellular+behavior+and+micromolecular+communication+in+urothelial+micrografts.">Insights into cellular behavior and micromolecular communication in urothelial micrografts.</a> Sci Rep. 13 (1), 13589 (2023).</li><li>Reinfeldt Engberg, G., Chamorro, C. I., Nordenskj&#246;ld, A., Fossum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+submucosal+bladder+wall+tissue+in+vitro+and+in+vivo.">Expansion of submucosal bladder wall tissue in vitro and in vivo.</a> Biomed Res Int. 2016, 5415012 (2016).</li><li>Juul, N., Ajalloueian, F., Willacy, O., Chamorro, C. I., Fossum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+autologous+urothelial+micrografting+and+composite+tubular+grafts+for+future+single-staged+urogenital+reconstructions.">Advancing autologous urothelial micrografting and composite tubular grafts for future single-staged urogenital reconstructions.</a> Sci Rep. 13 (1), 15584 (2023).</li><li>Willacy, O., Juul, N., Taouzlak, L., Chamorro, C. I., Ajallouiean, F., Fossum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perioperative+layered+autologous+tissue+expansion+graft+for+hollow+organ+repair.">A perioperative layered autologous tissue expansion graft for hollow organ repair.</a> Heliyon. 10 (3), e25275 (2024).</li><li>Chamorro, C. I., 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=Exploring+the+concept+of+in+vivo+guided+tissue+engineering+by+a+single-stage+surgical+procedure+in+a+rodent+model.">Exploring the concept of in vivo guided tissue engineering by a single-stage surgical procedure in a rodent model.</a> Int J Mol Sci. 23 (20), 12703 (2022).</li><li>Casarin, M., Morlacco, A., Dal Moro, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+substitution:+The+role+of+tissue+engineering+and+biomaterials.">Bladder substitution: The role of tissue engineering and biomaterials.</a> Process. 9 (9), 1643 (2021).</li></ol>

The authors have no conflicts of interest to declare.

This protocol presents a simple and approachable technique for future reconstructive surgeries. A common drawback in tissue engineering, including autologous cell expansion, is the expensive and substantial prefatory steps required before surgical implantation. Autologous micrografting may simplify many of these steps and potentially allow for single-staged procedures. By auto-transplanting complex histological entities, pro-regenerative paracrine signaling is induced18. In previous studies, we experienced that micrografts alone are vulnerable to the physical environments unless suitably attached to a scaffold15,19. Collagen has been studied as a viable environment for tissue expansion in vitro and was chosen for our purpose due to its favorable biocompatibility and commercial availability. The composite scaffold presented here has previously been optimized during in vitro experiments evaluating variations in micrograft embedding and collagen concentrations20,21,22. Before in vivo testing, the scaffold properties regarding permeability, biomechanics, and degradation have been evaluated in vitro20. Furthermore, the in vivo scaffold-based tissue expansion was previously validated in rodent and rabbit models21,22.
The surgical model was chosen to evaluate a tubular version of the scaffold, mimicking the clinical setting of a urinary diversion for neurogenic bladder dysfunction in pediatric or adolescent patients. The critical steps include the exact dissection of the mucosal micrografts and maintaining a moist environment from the time of resection to the scaffold embedding. Another critical step includes proper hydrogel solidification; careful pipetting of the collagen ensures that air bubbles are not formed within the gel, and correct temperature settings and component solutions ensure that the gel properly solidifies. Failure to obtain a solidified gel will increase the risk of collagen delamination and micrograft detachment. For the surgical part, careful handling during implantation is crucial to avoid damaging the micrografts due to mechanical trauma or dissociation. Before closing the abdomen, fluid patency should be carefully addressed by insufflating the bladder with fluids.
Limitations to the technique include the thickness of the scaffold, which intuitively has upper limits regarding the diffusion of nutrients from the external environment to the micrografts. On the other hand, a reduction in scaffold thickness may lead to inappropriately high permeability and urine leakage. Our current composition is based on previous in vitro assessments, where cell regeneration in varying collagen concentrations was compared20. Micrografting of autologous tissues also relies on healthy graft tissue, making the current procedure unsuited for malignant diseases where the risk of cancerous re-transplantation cannot be properly ruled out23; nevertheless, the current technique was designed for cases with functional voiding disabilities where this is not considered a risk. Although the model mimics several steps from the clinical setting (i.e., the appendicovesicostomy procedure), this experiment does not utilize a fully functional stoma for urinary diversion since the conduit is ligated distally. Also, as clinical complications can occur life-long, a 6 week observational period may provide limited knowledge on specific outcomes on strictures and continency. Therefore, an additional 6-month followup could be added to the study after anastomosing the healed conduit to the skin level.
The perspective of this technique relates to the simple design, enabling universal applications in case the micrograft tissue-origin and supporting biomaterial is replaced with other relevant alternatives. These components can be modified to suit organ-specific purposes related to scaffold strength, elasticity, and biodegradation. Finally, the accessible and low-cost expenses allow for reproducibility and a broadened translation of the technique.

In urogenital malformations, reconstructive surgery can be required to restore functional anatomy, often on a vital indication1,2. Conventional surgical approaches have utilized native tissues from other organ systems (such as the gastrointestinal tract) to reconstruct the malformed or missing organs; however, often with the risk of severe postoperative complications3,4. In the case of urinary diversion for patients with neurogenic bladder dysfunction in need of long-term catheterization, the appendix or re-tailored small bowel segments are often used to construct a urinary conduit5,6. Tissue engineering offers an alternative grafting tissue that can be tailored to meet organ-specific characteristics, thereby minimizing postoperative morbidity for the patients7,8. Whereas scaffolds of various kinds can be implanted on their own, additional scaffold cellularization, preferably with autologous cells, has been shown to improve the regenerative outcomes after implantation9,10,11,12,13,14. Nevertheless, tissue-engineered scaffolds are often comprised of complex and costly components, and secondly, the requirements for ex vivo cell culturing and scaffold seeding are laborious and resource-intensive. These factors have hindered the clinical translation of tissue-engineered scaffolds despite several decades of research within the area. By reducing the complexity as well as monetary and materialistic requirements, tissue-engineered scaffolds could be implemented in modern surgery on a broad scale, addressing both rare and more common procedures.
Collagen has previously been established as a viable platform for cell expansion and, furthermore, acts as a favorable bio-adhesive when attaching cells or tissue onto a scaffold for surgical implantation15,16,17. Perioperative autologous micrografting circumvents the need for ex vivo cell culturing by harvesting the tissue of interest during the primary procedure and re-implanting it directly. By mincing the resected tissue into smaller particles, the surface area and the growth potential is increased, allowing for a larger expansion ratio onto the scaffold18. The collagen-based scaffold does not adhere specifically to urogenital reconstructions but can theoretically apply to multiple areas of hollow-organ reconstruction.
In this manuscript, we present both a protocol for the construction of a tubular scaffold, combining collagen with embedded autologous urothelial micrografts, and a minipig model evaluating the technical feasibility and safety, as well as the regenerative performance, of the scaffold in vivo. The model was evaluated in 10 full-grown female minipigs using the protocol and method presented here. The main advantage of the scaffold is the simplicity of the construct and the single-staged implantation, sparing the patient of several subsequent surgical procedures. The procedure can be performed in conventional surgical settings by regular surgical personnel and requires standard equipment and materials. The animal model allows for a controlled environment for studying the implantation while the animal readily returns to normal behavior, with the added possibility of implementing variations to the scaffold and the procedure.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x MEM</td>
			<td>Gibco, Thermo Fisher Scientific, Waltham, US</td>
			<td>2517592</td>
			<td>Collagen preparation</td>
		</tr>
		<tr>
			<td>1x MEM</td>
			<td>Gibco, Thermo Fisher Scientific, Waltham, US</td>
			<td>2508924</td>
			<td>Collagen preparation</td>
		</tr>
		<tr>
			<td>Ambu aScope 4 Cysto</td>
			<td>Ambu A/S, Ballerup, DK</td>
			<td>1000682507</td>
			<td>Cystoscope</td>
		</tr>
		<tr>
			<td>Aquaflush ACE stopper</td>
			<td>Abena, Taastrup, DK</td>
			<td>ACE12/220501</td>
			<td>ACE stopper</td>
		</tr>
		<tr>
			<td>Borgal vet inj opl 200 + 40 mg/mL</td>
			<td>Ceva Animal Health A/S</td>
			<td>510460</td>
			<td>Sulfonamide/Trimethoprim</td>
		</tr>
		<tr>
			<td>Bupaq multidose vet 0.3 mg/mL</td>
			<td>Salfarm Danmark A/S, DK</td>
			<td>502763</td>
			<td>Buprenorphin</td>
		</tr>
		<tr>
			<td>Butomidor vet inj 10 mg/mL</td>
			<td>Salfarm Danmark A/S, DK</td>
			<td>531943</td>
			<td>Buthorphanol</td>
		</tr>
		<tr>
			<td>Comfortan vet inj 10 mg/mL</td>
			<td>Dechra Veterinary Products A/S, DK</td>
			<td>492312</td>
			<td>Metadone</td>
		</tr>
		<tr>
			<td>Ethilon suture 3-0</td>
			<td>Ethicon, Johnson &#38; Johnson, New Brunswick, US</td>
			<td>SGBCXV</td>
			<td>Monofilament non-resorbable</td>
		</tr>
		<tr>
			<td>Fentanyl inj 50 &#181;g/mL(hamel)</td>
			<td>Hameln Pharma ApS, DK</td>
			<td>432520</td>
			<td>Fentanyl</td>
		</tr>
		<tr>
			<td>Ketador vet inj 100 mg/mL</td>
			<td>Salfarm Danmark A/S, DK</td>
			<td>115727</td>
			<td>Ketamine</td>
		</tr>
		<tr>
			<td>Metacam inj 20 mg/mL t.cattle/pig/horse</td>
			<td>Boehringer Ingelheim Animal, DE</td>
			<td>6443</td>
			<td>Meloxcicam</td>
		</tr>
		<tr>
			<td>Metacam oral suspension 15 mg/mL pigs</td>
			<td>Boehringer Ingelheim Animal, DE</td>
			<td>482780</td>
			<td>Meloxcicam</td>
		</tr>
		<tr>
			<td>Omnipaque</td>
			<td>GF Healthcare, Oslo, NO</td>
			<td>16173849</td>
			<td>Contrast for CT</td>
		</tr>
		<tr>
			<td>Pancytokeratin CK-AE</td>
			<td>DAKO Agilent, US</td>
			<td>GA053</td>
			<td>Clone AE1/AE3</td>
		</tr>
		<tr>
			<td>PDS suture 3-0</td>
			<td>Ethicon, Johnson &#38; Johnson, New Brunswick, US</td>
			<td>SEMMTQ</td>
			<td>Monofilament slow-resorbable</td>
		</tr>
		<tr>
			<td>Prolene suture 4-0</td>
			<td>Ethicon, Johnson &#38; Johnson, New Brunswick, US</td>
			<td>PGH187</td>
			<td>Monofilament non-resorbable</td>
		</tr>
		<tr>
			<td>Propolipid t.inj/inf 10 mg/mL</td>
			<td>Fresenius Kabi, DK</td>
			<td>21636</td>
			<td>Propofol</td>
		</tr>
		<tr>
			<td>Rat-tail collagen type I</td>
			<td>First Link Ltd, Wolverhampton, UK</td>
			<td>60-30-810</td>
			<td>2.06 mg/mL protein in 0.6% acetic acid</td>
		</tr>
		<tr>
			<td>Suprim vet&#160; 20 + 100 mg (Solution for use in drinking water)</td>
			<td>Dechra Veterinary Products A/S, DK</td>
			<td>33661</td>
			<td>Sulfonamide/Trimethoprim</td>
		</tr>
		<tr>
			<td>SX-ELLA Degradable Biliary DV stent</td>
			<td>ELLA-CS, Trebes, CZ</td>
			<td>S23000056-01</td>
			<td>&#248; 6 mm x 60 mm</td>
		</tr>
		<tr>
			<td>Vicryl mesh</td>
			<td>Ethicon, Johnson &#38; Johnson, New Brunswick, US</td>
			<td>VM1208</td>
			<td>Mesh</td>
		</tr>
		<tr>
			<td>Vicryl suture 4-0</td>
			<td>Ethicon, Johnson &#38; Johnson, New Brunswick, US</td>
			<td>SMBDGDR0</td>
			<td>Braided fast-resorbable</td>
		</tr>
		<tr>
			<td>Xysol vet inj 20 mg/mL</td>
			<td>ScanVet Animal Health A/S, DK</td>
			<td>54899</td>
			<td>Xylazine</td>
		</tr>
		<tr>
			<td>Zoletil 50 vet plv/sol t.inj 25 + 25 mg/mL</td>
			<td>Virbac Danmark A/S, DK</td>
			<td>568527</td>
			<td>Tiletamine and Zolazepam</td>
		</tr>
	</tbody>
</table>

surgical model for single-staged tissue-engineered urothelial tubes in minipigs

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has been harvesting gastrointestinal tissue for non-orthotopic reconstruction due to its abundance to reestablish normal function in the patient. The clinical outcomes after rearranging native tissues within the body are often associated with significant morbidity; thus, tissue engineering holds specific potential within this field of surgery. Despite substantial advances, tissue-engineered scaffolds have not yet been established as a valid surgical treatment alternative, mainly due to the costly and complex requirements of materials, production, and implantation. In this protocol, we present a simple and accessible collagen-based tubular scaffold embedded with autologous organ-specific tissue particles, designed as a conduit for urinary diversion. The scaffold is constructed during the primary surgical procedure, comprises commonly available surgical materials, and requires conventional surgical skills. Secondly, the protocol describes an animal model designed to evaluate the short-term in vivo outcomes post-implantation, with the possibility of additional variations to the procedure. This publication aims to demonstrate the procedure step-by-step, with special attention to the use of autologous tissue and a tubular form.

In this study, in vivo urothelial tissue expansion is achieved in a collagen-based tubular scaffold. By embedding the scaffold with autologous tissue particles, harvested and processed perioperatively, the procedure allows for single-staged scaffold implantation without the need for concomitant immunosuppressive treatment postoperatively. Surgical handling is enabled by reinforcing the scaffold with a biodegradable mesh and stent (Figure 1). After 6 weeks of observation, the macroscopical tissue evaluation revealed no signs of host rejection or infection, and the tubular scaffold presents patent and unobstructed (Figure 2). From histological evaluations, a stratified luminal epithelium of urothelial origin is seen covering the entirety of the scaffold, and remnants of the reinforcing biomaterials are still visible after 6 weeks (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66936/66936fig01.jpg" /> 
Figure 1: Scaffold construction and implantation. The bladder tissue is dissected perioperatively (top left). The minced mucosal micrografts are expanded onto a surgical mesh (top middle) and embedded in solidified collagen (top right). The collagen has been compressed to expel water, and a stent is prepared (bottom left). The scaffold is tubularized around the stent, and an ACE stopper is placed inside the stent (bottom middle). The bladder is partially closed, and the construct is finally incorporated into the bladder at the original site of tissue excision (bottom right). <a href="https://www.jove.com/files/ftp_upload/66936/66936fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66936/66936fig02.jpg" /> 
Figure 2: Scaffold macroscopical evaluation. After 6 weeks, the animal is euthanized, and the scaffold (arrow) is dissected at the skin level (top left). The bladder is filled with contrast (yellow) and CT scanning is performed to evaluate the conduit (arrow) for patency and signs of stricture formation (top right). A cystoscopy is performed via the urethra to evaluate the bladder and the anastomosis (arrow) after 6 weeks (bottom left). The conduit is once more tested for patency by inserting a catheter (arrow) via the external opening and into the bladder (bottom right). <a href="https://www.jove.com/files/ftp_upload/66936/66936fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66936/66936fig03.jpg" /> 
Figure 3: Scaffold microscopical evaluation. The resected conduit is fixated, and orthogonal transverse sections are performed to evaluate the conduit in the proximal-distal direction. After 6 weeks, the conduit lumen (1) is evaluated to confirm epithelialization (magnified top). Remnants of the biodegradable stent (2) and mesh materials (magnified bottom) are still visible at this point. <a href="https://www.jove.com/files/ftp_upload/66936/66936fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Tissue-engineered implants for reconstructive surgery rarely progress beyond preclinical trials due to laborious ex vivo culturing, which includes complex and expensive scaffold components. Here, we present a single-staged procedure designed for urinary diversion with an accessible collagen-based tubular scaffold containing autologous micrografts.

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has ...

surgical-model-for-single-staged-tissue-engineered-urothelial-tubes

Research

JoVE Journal

Bioengineering

166 Views.  Copenhagen University Hospital - Rigshospitalet. Tissue-engineered implants for reconstructive surgery rarely progress beyond preclinical trials due to laborious ex vivo culturing, which includes complex and expensive scaffold components. Here, we present a single-staged procedure designed for urinary diversion with an accessible collagen-based tubular scaffold containing autologous micrografts.

Video: Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

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. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.