The authors would like to acknowledge UCL for funding this project.

NOTE: Experiment design and ethical approval were obtained from UCL Institutional Review Board (IRB). All experiments were carried out as per regulation of Home Office and guidelines of Animals (scientific procedure) Act 1986 with revised legislation of European Directive 2010/63/EU (2013). Rabbits were inspected by a named veterinary surgeon (NVS) periodically and twice in a day by a named animal care and welfare officer (NACWO) (As per guidelines and regulations of Home office). They did not show any sign of pain until they were euthanized.
1. Preparation of Tissue Engineered Tendon (ET) Graft
<ol>
	<li>To fabricate the collagen hydrogel, add 4 mL of rat tail collagen type 1 monomeric collagen solution (2.15 mg/mL in 0.6% acetic acid with 0.2% w/v of total protein) and 500 &#181;L of 10x Minimal Essential Medium. Neutralize this by titrating against 5 M and 1 M sodium hydroxide and add 500 &#181;L of Dulbecco&#39;s Modified Eagle Medium (DMEM).</li>
	<li>Pour 5 mL of this solution into a custom built rectangular metal mold (33 mm &#215; 22 mm &#215; 10 mm, 120 g weight) (Figure 1). Keep the mold in a CO2 incubator at 37 &#176;C and 5% CO2 for 15 minutes to allow matrix assembly19.</li>
</ol>
2. Fabrication of the Graft
<ol>
	<li>After polymerization, remove the collagen hydrogel from the mold and place in a standard plastic compression assembly (Figure 2A)19.</li>
	<li>Place the collagen hydrogel in between two 50 &#181;m nylon mesh sheets and apply a static load of 120 g (total surface area 7.4 cm2, which is a pressure equivalent to 1.6 kPa) for 5 minutes to remove interstitial fluid from the hydrogel (Figure 2A). Use four layers of filter paper to absorb the discharged fluid from hydrogels.</li>
	<li>Use four layers of compressed gels rolled on top of each other (Figure 2B) and cut into 15 mm segments (Figure 2C) to fabricate the ET. 
	NOTE: New Zeland white male rabbits of age 16 - 25 weeks were used in the experiments.</li>
	<li>Sedate animals with an intramuscular (i.m.) dose of Hypnorm (0.3 mg/mL) and euthanize by administering an overdose of pentobarbitone.</li>
	<li>Immediately after euthanasia, trim the hair on both hind legs. Then with a size 20 surgical blade, make a 9 cm incision around the inferior tibiofibular area to expose the tibialis posterior (TP) tendon.</li>
	<li>With the same sized surgical blade, excise lapine TP tendons with an average length of 70 mm and keep moist in PBS during the experimental process to avoid drying.</li>
</ol>
3. Developed Novel Tenorrhaphy Technique
NOTE: The sutures (see Table of Materials) are non-absorbable and made from an isotactic crystalline stereoisomer of polypropylene, which is a synthetic linear polyolefin. The core interlocking sutures were mainly consisting of 3-0 and the peripheral sutures were 6-0. These were the two main sutures used in all experiments.
<ol>
	<li>With a surgical blade, cut the TP tendon at the midpoint. Excise a 15 mm segment of the tendon from the middle of the tendon and replace it with the ET collagen graft (Figure 2D). Interlock the 3-0 suture proximally away from native tendon ends (Figure 3A).</li>
	<li>Pass the 3-0 core sutures above the entire length of the graft and interlock distally away from the cut end.</li>
	<li>Secure both ends of the ET to the native tendon with 6-0 and continuous running sutures around the periphery by coupling two tendon ends (Figure 3B). This is done so that the graft can be moved easily on the suture by placing tension on the native tendon20.</li>
	<li>After securing the suture as described above, manually ensure that the tension on the sutures is appropriate and that there is no flaccidity in the entirety of the suture.</li>
</ol>

The authors declare that they have no conflicts of interest.

In this study, tissue engineered type I collagen grafts was chosen as a tendon graft because collagen is a natural polymer and used as a biomaterial for various tissue engineering applications27,28. Also, collagen constitutes 60% of the dry mass of tendon, out of which 95% is type 1 collagen 21,29,30,31,32. For successful engraftment to occur, mechanical properties of the graft should ideally match the native tendon33; however, with current engineering techniques, the mechanical properties of ET (4.41 N) are significantly inferior to the native tendon (NT) (261.08 N)33. It is proposed that this is due to the highly organized hierarchical arrangement of collagen fibril in the native tendon, which remains a challenge to engineer and match its mechanical properties34. We have tried to increase the density of the ET matrix by applying a static weight of compression to the collagen hydrogel33; however, the architectural complexity from which the tendon acquires its strength is more intricate. Methods to accrue mechanical strength arguably are best attained in vivo, where the host biological processes can act on the remodelling of the extracellular matrix. Therefore, in this study, another strategy was adopted to modify the current suture technique as post tendon repair; the mechanical strength of the repaired tendon graft is entirely dependent on the suture technique8,9. Hence, by modifying existing suture techniques, we can offload the engineered tendon graft until cell and ECM induced remodelling occurs as a new approach.
To date, there are various suture techniques available to repair the tendon, none of which is a gold standard; however, the modified Kessler suture technique is widely used to repair tendons because it is less obstructive and damaging to tendons35,36. The flexor digitorum profundus muscle tendon of lambs, when sutured with the 6-strand Savage technique, was reported to have a break strength of 51.3 N, but when a modified Kessler suture technique was used, the break strength was 69.0 N7. However, in this study, when the tendon gap of 15 mm was filled with ET and repaired with Modified Kessler suture technique, the repair failed at an early stage with a break strength of 12.49 N (Figure 4). This low value makes the technique clinically irrelevant. Similar findings have been reported by De Wit et al. in a porcine flexor repair tendon model, suggesting that Kessler repair failed at suture rupture by reducing gapping by 15% as compared to cruciate repair, where gapping is reduced by 87% and repair failed at suture pull-out38. Thus, there is a need for another strong suture technique, which could hold mechanically weaker ET in place.
A novel modified suture technique was developed by using four core sutures over the entire length of the ET and above the opposite tendon. These sutures were interlocked onto the suture material itself at some distance away from each tendon end. This is mainly because it has been reported that putting suture knots at equal distance and equal load sharing tension on all suture strands increases their mechanical properties39. A balanced repair can also be achieved by keeping a continuous suture, and staggering the repair to allow for compression at the repair site40.
In this study, 3-0 sutures were used for outer interlocked sutures considering that rabbit TP tendon has a length, width and thickness of 62.4 mm, 5 mm and 1.5 mm, respectively. 6-0 sutures were used to hold the ET in place. Although we have tried other absorbable suture materials, it would not be appropriate as they become weaker over a period in vivo41. A primary reason polypropylene sutures was selected is because they are a monofilament as well as non-absorbable and they do not cause structural or tensional modifications under load42. We tested all sutures from 2-0 to 7-0, but 3-0 and 6-0 were found to be ideal candidates for our experiments 26.
The primary reason for using 4 strand repair was to avoid excessive damage to ruptured tendon ends with a greater number of suture strands as it has been reported that a normal surgical suture in a tendon results in the formation of an acellular region43. It has been hypothesized that this is due to the cells migrating out from the compressive load that is put on the tendon, and normally these cells are subject to tensile loading17. This migration of cells away from the suture could then cause weakening of the matrix as there is a paucity of cells to maintain and turnover the matrix, which could cause early tendon failure17. We can use more strands of sutures that are biomechanically twice as strong (ex vivo) than 4-strand sutures11,12,44,45; however, these repairs are not widely practiced and their clinical limitations are currently being evaluated13,14,15,16.
The placement of the suture knot is important but there are arguments for and against externalizing the suture. Having the suture on the outer surface can potentially snag against structures like tendon pulleys and reduce glide. In a study, the areas where suture knots are placed inside illustrated a decrease in gliding resistance compared with the Kessler repair, which has suture knots outside46. Studies conducted in the canine model concluded that at a higher magnitude of the force, fewer suture knots located outside the repair and away from the tendon ends had survived compared with those located inside the repair47,48. However, internalizing the knot potentially reduces the contact surface of the healing tendon. There is also the consideration that tissue damage arises from the suture needle piercing the tendon and the greater number of passes relates to the increased tendon trauma49.
To secure ET in between the tendon gap, a standard of running sutures50 along the edge of the tendon and ET was performed. This was done because there was a need for peripheral sutures that are strong enough to hold the ET in place in the initial phase of healing until cell and ECM induced remodelling could occur50. The major problem was the variation in the mechanical properties of the NT and ET, which could result in early gap formation although the ET was stress shielded. On the other hand, applying a more secure technique such as horizontal mattress intrafiber sutures51, Halsted continuous horizontal mattress sutures52,53, cross stitch epitendinous repair techniques54,55,56,57 or running lock sutures58,59 would have ruptured ET as it is fragile. Thus, we chose running sutures as a peripheral suture technique which is simple and holds the ET intact in all directions.
From a tissue engineering perspective, we need to study whether this method can be used to fill a tendon gap greater than 1.5 cm. To use this graft in human clinical trials, we need to further investigate the immunological response to the xenogeneic source of collagen although this can be achieved by developing clinical grade collagen. The protocol described herein establishes the feasibility of the developed suture technique within available anatomical spaces in a porcine lapine model. This developed suture technique has suture points proximally and distally equidistance away from ruptured tendon ends so that engineered tendon graft could be off loaded. Hence, it could mature and integrate in vivo.

Tendon rupture may occur due to extrinsic factors such as traumatic lacerations or excessive loading of the tendon. Due to the external tensile forces placed on a tendon repair, a gap inevitably forms with most tendon repair techniques. Currently, tendon defects/gaps are filled with auto-, allo-, xeno- or artificial grafts, but their availability is finite, and the donor site is a source of morbidity.
The tissue-engineered approach to fabricate tendon graft from a natural polymer such as collagen has the distinctive advantage of being biocompatible and can provide vital extracellular matrix (ECM) components that facilitate cell integration. However, due to a lack of fibrillar alignment, the mechanical properties of the engineered tendon (ET) are inferior to the native tendon. To increase mechanical properties of the weaker collagen, many methods have been used, such as physical cross-linking under vacuum, UV radiation, and dehydrothermal treatments1. Also, through chemical cross-linking with riboflavin, enzymatic and non-enzymatic methods increased collagen density and the Young&#39;s modulus of the collagen in vitro2,3. However, by adding cross-linking agents, biocompatibility of the collagen is compromised, as studies have shown a 33% alteration in mechanical properties and 40% loss of cell viability3,4,5. Gradual accruement of alignment and mechanical strength can be obtained through cyclic loading6; however, this can be efficiently acquired in vivo7.
For ET to integrate in vivo and acquire strength without the need for chemical alteration, one approach would be to use a stabilizing suture technique to hold the weaker construct in place. Most tendon repairs rely on the suture design to hold tendon ends together; hence modification of these existing techniques could provide a logical solution8,9.
Until the 1980s, 2-strand repairs were widely used, but recent surgical literature describes the use of 4 strands, 6 strands or even 8 strands in repair10,11. In 1985, Savage described 6-strand suture techniques with 6 anchor points, and it was significantly stronger than the Bunnell suture technique that uses 4 strands 12. Also, 8-strand repairs are 43% stronger than other strands in cadaver and in situ models, but these repairs are not widely practiced as it becomes technically difficult to reproduce the repairs accurately13,14,15,16. Therefore, a greater number of core suture strands relates to a proportional increase in biomechanical properties of the repaired tendon. However, there is a loss of cell viability around the suture points, and trauma from excessive suturing can be to the detriment of the tendon, which can compromise tendon healing17. Suture techniques should provide a strong geometric repair that is balanced and relatively inelastic to minimize tendon gapping after repair. In addition, the location of the suture and its knots have to be strategically placed in order for them not to interfere with gliding, blood supply and healing until accruement of adequate strength has been obtained10,18.
To establish feasibility to secure weaker ET graft or other graft material in between ruptured tendon, we have developed a novel suture technique that can offload the graft so that it can mature and gradually integrate into the host tissue in vivo.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rat tail type 1 Collagen&#160;</td>
			<td style="width:271px;">First Link, Birmingham, UK</td>
			<td style="width:119px;">60-30-810</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">prolene sutures 6-0</td>
			<td>Ethicon Ltd, Edinburgh, U.K.</td>
			<td>EP8726H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">prolene sutures 3-0</td>
			<td>Ethicon Ltd, Edinburgh, U.K.</td>
			<td>D8911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Whatman filter paper</td>
			<td style="width:271px;">SIGMA-ALDRICH&#160;</td>
			<td style="width:119px;">WHA10010155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco&#160;DMEM, high glucose</td>
			<td style="width:271px;">Thermo Fisher Scientific&#160;</td>
			<td>11574486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nylon mesh&#160;</td>
			<td>Plastok (Meshes and Filtration) Ltd.</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We have used collagen grafts fabricated from type I collagen, as this is the predominant protein found in the tendon. It constitutes almost 95% of total collagen in the tendon; hence, collagen has exhibited all ideal properties for mimicking tendon in vivo21,22.
In this study, the type I collagen used was extracted from rat tail tendon and dissolved in the acetic acid (2.15 mg/mL). To polymerize this collagen, it was neutralized with sodium hydroxide in vitro, which formed non-cross-linked anisotropic collagen fibrils. This hydrogel contains 98% fluid and could mimic living tissue in vivo within 20 minutes during fabrication23. However, this hydrogel is mechanically weak; therefore, to increase mechanical properties, we have developed a method for rapid compression of collagen hydrogel by a technique known as &#39;plastic compression&#39;, where the degree of compression is directly proportional to the applied weight on the top and released fluid from the fluid leaving surface (FLS)19.
Spiral rolling of this graft increases its mechanical properties19, but the graft remains significantly weaker than the native tendon. To address this issue, we have developed a novel modified suture technique by placing suture points, not at the edge of ruptured tendons but proximally and distally away. Thus, the strength of the repair is on the sutures and suture points and not on the mechanically weaker tendon graft.
To demonstrate the functionality of the developed novel suture technique, a lapine TP tendon was excised. The gap was filled with a 15 mm long tendon graft secured with 6-0 sutures, and 3-0 interlocked sutures were placed at 70 mm to act as load barriers (Figure 3A). The mean break strength of repair was 50.62 &#177; 8.17 N, which was significantly higher (p &#60; 0.05) than that of the control Kessler repair of 12.49 &#177; 1.62 N (Figure 4A). Hence, core suture length and their interlocking away from the tendon ends significantly influence resistance of the tendon and the repairs from failing at higher magnitude forces24,25.
This resistance was inadequate in the control repairs which caused early repair failure and strain failure of more than 20% on the tendon.However, this is a physiological anomaly, as tendons in vivo are never subject to 20% straindue to there not being enough space for a tendon to extend that much; therefore to test feasibility of the suture technique in vivo models, we have performed repair in situ and calculated a mean break strength of 24.60 &#177; 3.92 N, which is significantly higher than the control mean break strength of 13.98 &#177; 2.26 N (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57696/57696fig1.jpg" /> 
Figure 1: Neutralized collagen hydrogel (pH 7.4) (pink color) cast in the stainless steel mold. Gel was allowed to remain in a CO2 incubator at 37 &#176;C for 20 minutes for fibrillogenesis to occur. The scale bar is shown at the bottom. <a href="https://www.jove.com/files/ftp_upload/57696/57696fig1large.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/57696/57696fig2.jpg" /> 
Figure 2: Plastic compression process.&#160;(A) The collagen hydrogel placed in between nylon meshes with a constant static load of 120 grams applied. Drained fluid was absorbed by four layers of filter paper. The arrow shows the fluid leaving surface (FLS) for the gel. (B) Four layers of compressed collagen sheets were rolled along the axis to form &#39;engineered tendon&#39; (ET). (C) The section of ET was cut into 15 mm segments to mimic tendon. (D) The tendon defect was created in the native tendon (NT) by excising a 15 mm segment of the posterior tibial tendon, and the defect was filled with ET. This panel was modified from previous work26. <a href="https://www.jove.com/files/ftp_upload/57696/57696fig2large.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/57696/57696fig3.jpg" /> 
Figure 3: (A) Tendon defect was filled with ET and secured with 6-0 sutures, and the 3-0 interlocking four strand suture technique was performed passing above graft in the 30 mm region. Block arrow shows the starting point for the suture and the blank arrow shows the end point of the suture. This panel was modified from previous work26. (B) Feasibility of performing developed suture technique in a space inside lapine model (in situ). <a href="https://www.jove.com/files/ftp_upload/57696/57696fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57696/57696fig4.jpg" /> 
Figure 4: Mechanical strength. (A) A mechanical test output of the repair and (B) in situ mechanical test output (Error bars = SD; *p &#60; 0.05, one-way ANOVA with Bonferroni correction). This panel was modified from previous work26. <a href="https://www.jove.com/files/ftp_upload/57696/57696fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects at JoVE.com

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

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.

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

a-novel-tenorrhaphy-suture-technique-with-tissue-engineered-collagen

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.8K Views.  University College London. 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.

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

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.

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Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

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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.

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

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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

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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.

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Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...