Name: a novel tenorrhaphy suture technique with tissue engineered collagen graft to repair large tendon defects
Uploaded: 2021-12-10T13:00:00.000+00:00
Duration: 6 min 36 s
Description: Watch this Scientific Journal Video about A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects at JoVE.com

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.

<table><tbody><tr><td>Rat tail type 1 Collagen&amp;nbsp;</td><td>First Link, Birmingham, UK</td><td>60-30-810</td><td></td></tr><tr><td>prolene sutures 6-0</td><td>Ethicon Ltd, Edinburgh, U.K.</td><td>EP8726H</td><td></td></tr><tr><td>prolene sutures 3-0</td><td>Ethicon Ltd, Edinburgh, U.K.</td><td>D8911</td><td></td></tr><tr><td>Whatman filter paper</td><td>SIGMA-ALDRICH&amp;nbsp;</td><td>WHA10010155</td><td></td></tr><tr><td>Gibco&amp;nbsp;DMEM, high glucose</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>11574486</td><td></td></tr><tr><td>Nylon mesh&amp;nbsp;</td><td>Plastok (Meshes and Filtration) Ltd.</td><td>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 'plastic compression', 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 ± 8.17 N, which was significantly higher (p &lt; 0.05) than that of the control Kessler repair of 12.49 ± 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 ± 3.92 N, which is significantly higher than the control mean break strength of 13.98 ± 2.26 N (Figure 4B).
<img alt="Collagen hydrogel formation in stainless steel mold, labeled diagram for biomaterials research." class="xfigimg" src="/files/ftp_upload/57696/57696fig1.jpg" data-altupdatedat="1747929244000" data-alt="Figure 1"> 
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 °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="Tendon engineering process with hydrogel compression, collagen sheet rolling, and result comparison." class="xfigimg" src="/files/ftp_upload/57696/57696fig2.jpg" data-altupdatedat="1747929244000" data-alt="Figure 2"> 
Figure 2: Plastic compression process. (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 'engineered tendon' (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="Surgical suture technique diagram and result; tendon repair with modified grasp, running sutures." class="xfigimg" src="/files/ftp_upload/57696/57696fig3.jpg" data-altupdatedat="1747929244000" data-alt="Figure 3"> 
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="Bar chart comparing tendon repair force: control vs. modified repair vs. native tendon." class="xfigimg" src="/files/ftp_upload/57696/57696fig4.jpg" data-altupdatedat="1747929244000" data-alt="Figure 4"> 
Figure 4: Mechanical strength. (A) A mechanical test output of the repair and (B) in situ mechanical test output (Error bars = SD; *p &lt; 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

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

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

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

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

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

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 ...

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

Using Q Suture to Enhance Resistance to Gap Formation and Tensile Strength of Repaired Flexor Tendons

Peripheral epitendinous sutures are believed to enhance core suture strength in tendon repair and decrease the risk of gapping between tendon ends. Here Q suture, an alternative to peripheral sutures, is presented for the use in tendon repair. Its effects on gap formation and tensile strength of the repaired tendons were compared with conventional running peripheral sutures. Three 2-strand sutures and three 4-strand sutures were used in repairing porcine tendons. The time required for performing 2Q and running sutures were recorded. The repaired tendons were subjected to a cyclic loading test, and the cycle number, during which a 2-mm gap was formed, was determined. After the cyclic loading, the gap size at the tendon ends and the ultimate strength of the repaired tendons were measured. Augmentation with the Q sutures reduced the number of tendons showing 2-mm gaps at tendon ends during cyclic loading. With addition of Q sutures 2-strand sutures significantly increased the ultimate strength of the repaired tendons and 4-strand sutures decreased the gap distance at the repair site of tendons. The time required for performing 2Q sutures was significantly less than that for running sutures. Therefore, we conclude that the Q suture is efficient in enhancing the tensile resistance and tendon repair strength and can be an alternative to conventional peripheral sutures.

Here, we present a &#8220;Q&#8221; suture technique that can be performed in tendon repair and its effects on the gap formation and tensile strength of the repaired tendons. Q suture is shown to be efficient in enhancing the tensile resistance and tendon repair strength.

Peripheral epitendinous sutures are believed to enhance core suture strength in tendon repair and decrease the risk of gapping between tendon ends. Here Q ...

Medicine

Polytetrafluoroethylene (PTFE) as a Suture Material in Tendon Surgery

With the evolution of suture materials, there has been a change in paradigms in primary and secondary tendon repair. Improved mechanical properties allow more aggressive rehabilitation and earlier recovery. However, for the repair to hold against higher mechanical demands, more advanced suturing and knotting techniques must be assessed in combination with those materials. In this protocol, the use of polytetrafluoroethylene (PTFE) as a suture material in combination with different repair techniques was investigated. In the first part of the protocol, both linear tension strength and elongation of knotted against not-knotted strands of three different materials used in flexor tendon repair were evaluated. The three different materials are polypropylene (PPL), ultra-high molecular weight polyethylene with a braided jacket of polyester (UHMWPE), and polytetrafluoroethylene (PTFE). In the next part (ex vivo experiments with cadaveric flexor tendons), the behavior of PTFE using different suture techniques was assessed and compared with PPL and UHMWPE.
This experiment is comprised of four steps: harvesting of the flexor tendons from fresh cadaveric hands, transection of the tendons in a standardized manner, tendon repair by four different techniques, mounting, and measurement of the tendon repairs on a standard linear dynamometer. The UHMWPE and PTFE showed comparable mechanical properties and were significantly superior to PPL in terms of linear traction strength. Repairs with four- and six-strand techniques proved stronger than two-strand techniques. Handling and knotting of PTFE are a challenge due to very low surface friction but fastening of the four- or six-strand repair is comparatively easy to achieve. Surgeons routinely use PTFE suture material in cardiovascular surgery and breast surgery. The PTFE strands are suitable for use in tendon surgery, providing a robust tendon repair so that early active motion regimens for rehabilitation can be applied.

Polytetrafluoroethylene PTFE as a Suture Material in Tendon Surgery

The present protocol illustrates a method for assessing the biophysical properties of tendon repairs ex vivo. A polytetrafluoroethylene (PTFE) suture material was evaluated by this method and compared to other materials under different conditions.

With the evolution of suture materials, there has been a change in paradigms in primary and secondary tendon repair. Improved mechanical properties allow ...

Techniques for Lower Trapezius Transfer Using Achilles Allograft in Irreparable Posterosuperior Rotator Cuff Tears

The management of irreparable rotator cuff tears presents significant challenges, particularly in active individuals experiencing functional limitations, such as reduced forward elevation and deficits in both external and internal rotation. Traditional latissimus dorsi (LD) tendon transfer has shown effectiveness in reducing pain associated with posterosuperior cuff tears but often yields inconsistent functional outcomes. This is largely due to the LD's primary role as an internal rotator, which limits its capacity to restore normal shoulder biomechanics. To address these limitations, the lower trapezius (LT) tendon transfer, augmented with an Achilles allograft, has emerged as an alternative to enhance external rotation, leveraging the LT's line of pull, which closely resembles that of the infraspinatus muscle.
This protocol outlines a modified surgical technique for LT tendon transfer with Achilles allograft augmentation, detailing patient positioning, tendon harvest, graft preparation, arthroscopic passage, and fixation methods. The protocol emphasizes key anatomical landmarks to minimize neurovascular injury and enhance graft integration. Postoperative care includes a 3 month immobilization period followed by a structured rehabilitation program to facilitate functional recovery.
This procedure is indicated for a specific patient group requiring improved external rotation and is biomechanically advantageous over the LD transfer. Though additional studies are warranted to confirm its efficacy in broader patient populations, early clinical outcomes suggest that LT transfer with Achilles allograft could offer superior biomechanical alignment and improved external rotation.

Here, we describe a modified technique for lower trapezius tendon transfer using an Achilles allograft in the treatment of massive posterosuperior rotator cuff tears.

The management of irreparable rotator cuff tears presents significant challenges, particularly in active individuals experiencing functional limitations, ...

Dermal Allograft and Biceps Superior Capsule Reconstruction for Massive Irreparable Rotator Cuff Tears

Since the use of autologous fascia lata for superior capsule reconstruction of massive irreparable rotator cuff tears (MIRCTs), the technique has evolved into various modifications, including dermal allografts, long head of the biceps tendon (LHBT), and combinations of both, which will be discussed in this article. After making sure the remnant cuff cannot be restored to the anatomical footprint of the supraspinatus, a double-loaded or triple-loaded, suture-based anchor is inserted 5-8 mm posterior to the bicipital groove to secure the long head of the biceps (LHBT) initially. A bone trough is made 5 mm posterior to the bicipital groove. One or two lasso loops are created through the LHBT before the complete release of the transverse humeral ligament without tenotomy of the LHBT distal to the fixation point, resulting in a posteriorly rerouted LHBT.Preservation of the proximal attachment of the biceps on the glenoid side is maintained, ensuring a native fixation. 
Subsequently, a 3 x 3 cm dermal allograft of 2 mm thickness is utilized to cover the rerouted LHBT, enhancing its strength and providing a tensile effect. Four anchors are then employed for fixation: two double-loaded anchors at the glenoid side and two lateral row anchors at the greater tuberosity. Following the introduction of the dermal allograft into the joint, the sutures from the glenoid anchors are secured, and the optimal tension of the allograft is gauged during the insertion of the lateral row anchors at 45&#176; shoulder abduction. The dermal allograft can cover the LHBT to increase the spacer effect. Medial row anchors are not necessary. The remaining portions of the supraspinatus and infraspinatus can be repaired using sutures passed through the lateral row anchors or repaired with the dermal allograft together to enhance stability.

Here, we describe a modified technique for dermal allograft and long head of the biceps tendon superior capsule reconstruction for massive irreparable rotator cuff tears.

Since the use of autologous fascia lata for superior capsule reconstruction of massive irreparable rotator cuff tears (MIRCTs), the technique has evolved ...

Q Suture Technique: A Suturing Technique to Reduce Gap Formation and Increase Tensile Strength in Tendon Repair

To perform a tendon repair, begin by marking the anterior surface of one of the porcine tendon stems, with two points that are 10 millimeters from the cut tendon, one-fourth of the way from the left and right sides of the tendon in the medial-lateral direction. Mark each of the left and right lateral surfaces of the tendon with one point that is 8 millimeters from each cut tendon end, and locate the point in the middle of the anterior-posterior direction.
Then, use a vernier caliper to measure all of the lengths between each set of points. Using 4-0 sutures, insert a needle into the cut surface of one tendon stump from the middle point in the anterior-posterior direction, and one-fourth of the way from the left in the medial-lateral direction.
Draw a region of interest around the selected neuron, and draw a second larger region of interest around the tail that includes the first region of interest. Reinsert the needle obliquely from point three, and pass the needle transversely toward point four. Pull out the suture to create a small loop at the lateral surface of the tendon, and reinsert the needle obliquely from point two, passing the needle longitudinally toward the cut end.
Then, pull out the suture. To form a symmetrical repair, insert the needle into the cut end of the other tendon stump, and suture the other stump with the same construct. Tighten the suture with a 10% shortening of the tendon segment within the core suture, and tie the tendon ends together with three to four knots to complete the two-strand core suture.
To complete a four-strand core suture, repeat the suturing process as just demonstrated. To add Q suture, insert the same needle into the tendon anterior surface, 2 millimeters away from the joined tendon end, and withdraw the needle on the posterior surface of the tendon. Reinsert the needle into the posterior surface of the tendon, 2 millimeters away from the other side of the joined tendon end. Pull out the suture from the anterior surface of the tendon, and tie three knots to complete the first Q suture.
Then, repeat the procedure to complete the second Q suture. To add a running suture in the two-strand or four-strand core suture plus running group, use a 6-0 suture to add a running epitendinous suture of 9 to 10 stitches to the tendon ends, keeping a similar purchase of 1.5 millimeters and a depth of 1 millimeter. When all of the sutures have been placed, cover the repair of the tendon with wet gauze.