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

Summary

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.

Abstract

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.

Introduction

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's modulus of the collagen in vitro^2,3. 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 viability^3,4,5. 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 solution^8,9.

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 repair^10,11. 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 accurately^13,14,15,16. 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 obtained^10,18.

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.

Protocol

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

1. 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 µL of 10x Minimal Essential Medium. Neutralize this by titrating against 5 M and 1 M sodium hydroxide and add 500 µL of Dulbecco's Modified Eagle Medium (DMEM).
2. Pour 5 mL of this solution into a custom built rectangular metal mold (33 mm × 22 mm × 10 mm, 120 g weight) (Figure 1). Keep the mold in a CO₂ incubator at 37 °C and 5% CO₂ for 15 minutes to allow matrix assembly¹⁹.

2. Fabrication of the Graft

1. After polymerization, remove the collagen hydrogel from the mold and place in a standard plastic compression assembly (Figure 2A)¹⁹.
2. Place the collagen hydrogel in between two 50 µm 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.
3. 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.
4. Sedate animals with an intramuscular (i.m.) dose of Hypnorm (0.3 mg/mL) and euthanize by administering an overdose of pentobarbitone.
5. 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.
6. 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.

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.

1. 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).
2. Pass the 3-0 core sutures above the entire length of the graft and interlock distally away from the cut end.
3. 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.
4. 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.

Results

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 vivo^21,22.

In this study, the type I collagen used was extracted from rat tail tendon and dissolved in the aceti...

Discussion

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 applications^27,28_. Also, collagen constitutes 60% of the dry mass of tendon, out of which 95% is type 1 collagen ^21,29,30,31,

Materials

Name	Company	Catalog Number	Comments
Rat tail type 1 Collagen	First Link, Birmingham, UK	60-30-810
prolene sutures 6-0	Ethicon Ltd, Edinburgh, U.K.	EP8726H
prolene sutures 3-0	Ethicon Ltd, Edinburgh, U.K.	D8911
Whatman filter paper	SIGMA-ALDRICH	WHA10010155
Gibco DMEM, high glucose	Thermo Fisher Scientific	11574486
Nylon mesh	Plastok (Meshes and Filtration) Ltd.	NA