Substitut ligamentaire produit par génie tissulaire pour remplacer
le LCA rupturé: adaptations pour des applications cliniques.
Simon F.1*, Moreira Pereira J.1*, Lamontagne J.1, Cloutier R.1, Goulet F.2 and Chabaud S.1Institutions: 1CMDGT, Centre de Recherche du CHU de Québec-Université- Laval, Hôpital de l’Enfant-Jésus, 1401 18e
rue, Quebec, QC, Canada, G1J 1Z4. 1
Département de chirurgie and 2Département de réadaptation, Université Laval, Québec, QC, Canada, G1K 7P4.
Auteur de correspondance: Stéphane Chabaud, Ph.D. courriel: stefchabo@hotmail.com
*: Simon F. et Moreira Pereira J. doivent être considérés tous les deux premiers
auteurs sur cette publication.
RÉSUMÉ
Le ligament croisé antérieur (LCA) de l'articulation du genou, l'un des ligaments les plus forts du corps, est souvent la cible de lésions traumatiques. Malheureusement, son potentiel de guérison est limité et les options chirurgicales pour son remplacement sont fréquemment associées à des problèmes cliniques. Notre groupe de recherche a mis au point un premier substitut du LCA par génie tissulaire, doté de cellules autologues, qui a été greffé et intégré avec succès dans l’articulation du genou caprin (Goulet et coll, 2004). Des substituts acellulaires ont été greffés avec succès dans le modèle de chèvre. L'échafaudage naturel en collagène du substitut du LCA contribue à soutenir la migration, la croissance et la différenciation des cellules après l'implantation. Les bouchons osseux reliés par un fil chirurgical faisant partie de l’échafaudage des substitut du LCA permettent la méthode d’implantation os-ligament-os. Cependant, l’utilisation des os pose un problème lié à un manque potentiel de disponibilité des os. Ainsi, des modifications sont suggérées pour concevoir une deuxième génération de substituts du LCA, qui offrirait plus de polyvalence en tant que greffe biocompatible pour le remplacement du LCA déchiré chez l'humain.
Tissue-engineered ligament substitute for torn ACL replacement:
adaptations for clinical applications.
Simon F.1*, Moreira Pereira J.1*, Lamontagne J.1, Cloutier R.1, Goulet F.2 and
Chabaud S.1
Institutions: 1CMDGT, Centre de Recherche du CHU de Québec-Université- Laval, Hôpital de l’Enfant-Jésus, 1401 18e
rue, Quebec, QC, Canada, G1J 1Z4. 1
Département de chirurgie and 2Département de réadaptation, Université Laval,
Québec, QC, Canada, G1K 7P4.
Corresponding author: Stéphane Chabaud, Ph.D. email: stefchabo@hotmail.com
*: Simon F. and Moreira Pereira J. must be considered both as first author on this
publication.
ABSTRACT
The anterior cruciate ligament (ACL) of the knee joint, one of the strongest ligaments of the body, is often the target of traumatic injuries. Unfortunately, its healing potential is limited and the surgical options for its replacement are frequently associated with clinical issues. Our research group has developed a first bioengineered ACL (bACL) seeded with autologous cells, that was successfully grafted and integrated in goat knee joints. Acellular bACLs were grafted with success in the goat model. The natural collagen scaffold of the bACL contributes to support cell migration, growth and differentiation post-implantation. The bone plugs linked by a surgical suture that are part of the scaffold of the bACLs allow the bone-ligament-bone implantation method. However, the use of bones raise an issue associated to a potential lack of bone availability. Thus, modifications are suggested to design a second generation of bACL, that would offer more versatility as a biocompatible graft for torn ACL replacement in humans.
INTRODUCTION
The main function of anterior cruciate ligament (ACL) is to stabilize the knee. ACL tears are common injuries. Let the ACL heal by itself does not give good results because the formation of a fibrin clot is inhibited in the synovium leaving patients with an unstable knee ACL reconstruction could reduce the 10-years incidence of chronic instability of knee joints, meniscus and cartilage damage then degenerative osteoarthritis. 400,000 ACL reconstructions are performed each year worldwide, for an approximate cost of US$ 18 billons in USA. Nevertheless, current gold standard techniques suffer of a high rate of failure, total of about 12% especially in young patients. It could be also noted that the transplantation of tendon could result in lack of available tissues and risk of disease transmission for allografts and donor-site morbidity for autografts. For now, only 50 to 65% of injured people who undergo surgical reconstruction of ACL return to the same level of activity.
Tissue engineering has opened new and innovative approaches for the development of tissue substitutes and eventually reduce the needs in organ donors. Under ideal conditions, a reconstructed tissue should be easily integrated in the host post-grafting, to become permanently and efficiently integrated in situ. Thus, the matrix or scaffold of any reconstructed tissue must be strong enough to withstand physiological stresses early post-grafting and undergo remodeling to become functional. Since collagen is the natural component that supports most connective tissues, lyophilized collagen is an excellent choice to create biodegradable scaffolds (Goulet et al., 2004). The graft has to be colonized by surrounding cells which will reorganize its structural properties in response to the tensions and movements that it will support. Interestingly, collagen can be mixed with other matrix components and/or growth factors to stimulate cell migration, growth and differentiation.
A successful example of a biocompatible tissue engineered graft is our bACL that was integrated in goat knee joints. ACL bundles originate from the posteromedial aspect of lateral femoral condyle and insert over the tibia just anterior to the intercondylar eminence. The diameter of the bundles varies from 7 to 17 mm, while their length varies between 28 and 38 mm (Girgis et al., 1975; Odensten and Gillquist, 1985), depending on the individuals. The cross section of the ACL bundle in midsubstance cross section varies from 36 to 44 mm2 (Khatri et al., 2018). A collagen scaffold, anchored with two bone plugs and seeded with autologous ACL fibroblasts, was developed in vitro and grafted in a knee joint to replace torn ACL in vivo (Goulet et al., 2000, 2004, 2006, 2007 and 2011; Hart et al., 2005; Tremblay et al., 2011). An absorbable surgical thread was added to the collagen matrix, to contribute to its structural reinforcement and facilitate its manipulation during implantation (Goulet et al., 2004). Bone plugs were used to fix both ends of the graft, adding screws in the bone tunnels to fix the plugs. This approach was very successful in the goat model (Goulet et al., 2004). Such bACL grafted in situ reached an average of 36% (à 5%) of a native ACL strength after only 13 months, without any specific training program applied on the goats post-surgery. However, to simplify bACL’s production, the integration potential of acellular grafts was assessed on a group of 3 goats for 6 months. The results obtained on the goat model confirmed that such an approach was feasible to replace torn ACL. The acellular grafts became cellularized and reinforced in situ post-implantation. On the basis of these observations, further modifications were assessed in vitro. Despite the fact that the validity of the concept was confirmed in the caprine model, the matrix scaffold would still gain at being simplified. The use of bone anchors works very well, but it also creates a need for bone samples that may eventually limit the feasibility of the tissue engineering approach. The bACL would gain to be adapted to be eventually shipped and grafted in any surgical room, anywhere in the world. The bACL’s collagen scaffold remains fragile to shocks. Passing the graft through the tunnels performed in the tibial plateau and the femoral condyle causes wear. Thus, the bACL must be protected during its insertion in situ. We
report here the results of experiments that were designed to investigate the feasibility of these modifications, producing a second generation of graftable bACL.
MATERIALS AND METHODS
Preparation of acellular graftable bACLs
A group of 12 bACLs was prepared for this experiment. At least 3 bACLs would be used for histological analyses to insure quality control, before the surgical implantation of 3 other bACLs in the goat model. A number of 3 more bACLs was kept as backup in case something went wrong during the surgical procedure. The last 3 bACLs were subjected to mechanical rupture tests to measure their ultimate strength values before grafting. The methods that were developed to produce and graft bACLs in goat knee joints were reported previously (Goulet et al., 2004;
Tremblay et al., 2011). Briefly, to achieve the permanent fixation of the bACL to the bones, cylindrically shaped bovine bone plugs were prepared and pierced with a transverse hole. They were rinsed and stored in 100% ethanol for 2-3 days. A surgical thread (Maxon, size 3-0; Sherwood-Davis & Geck, St-Louis, MO), absorbable within 4-6 weeks post-surgery, was passed through the holes in the two bone plugs and tied. The bones and thread were counter-rotated to provide a single, twisted-thread link between the plugs. This bone/thread scaffolding was transferred to a sterile plastic tube and kept extended in a central, suspended position by passing two metal pins across the tube and through the transverse holes in the bone plugs (Goulet et al.; 2004; Tremblay et al., 2011).
For casting the bACLs, DMEM containing 10% FCS and 1.0 mg/ml of bovine type I collagen (isolated in our laboratory from healthy Canadian beef skin, tested for its purity by electrophoresis and solubilized in acetic acid diluted 1000 times with sterile water) was gently mixed. The mixture (total of 10 ml) was poured into 12-ml sterile plastic tubes containing the bone plugs linked by the surgical thread. The collagen polymerized in the mixture within 20 min at room temperature, under a sterile culture flow hood, while maintained without any agitation. Then, they were frozen in sterile petri dishes overnight at -70°C and subsequently lyophilized. They were transferred back into new sterile plastic tubes and fixed again with pins as
previously described. Following rehydration in fresh DMEM to produce a semi-rigid central core, a second coating of collagen was applied as described above. Bilayered bACLs were obtained with a lyophilized core and an outer layer. All bACLs were kept in DMEM supplemented with 10% FCS, 50 µg/ml ascorbic acid and antibiotics until the day of the surgical implantation.
Surgical procedures for implantation of acellular bACLs into 3 goats.
All surgical implantation procedures were performed under general anesthesia on 3 goats of 45 kg whose native ACLs were resected at the time of implantation of the bACLs (Goulet et al., 2004). Thus, a group of 3 acellular bACLs were grafted in 3 goat's knee joints. Only one leg of each goat was grafted, since the other was used as positive control.
Histological analysis of bACLs before and after implantation, ex-vivo.
Histological studies were performed on bACLs before implantation and at 6 months post-implantation. The bACL samples were fixed in an aldehyde-containing solution, embedded in paraffin, sectioned and stained by either Masson’s trichrome or Heamatoxylin-eosin methods to visualize the collagen matrix, the cells that had colonized the graft including endothelial cells in blood vessels and chondrocytes. The coloration of Holmes allowed the detection nerve endings in the grafts.
Mechanical analyses.
After 6 months, the bACLs will be dissected, keeping the femur-tibia system intact. All grafts and contralateral ACLs will be subjected to mechanical tests (ultimate strength and rigidity), using a testing machine (Instron, Corporation, Norwood, MA, USA).
Goat dermal fibroblasts.
Primary populations of goat dermal fibroblasts (DFs) were isolated previously from small biopsies of tissues, taken from several goats under anesthesia. The cells were extracted by enzymatic digestion (Goulet et al., 2004). The DFs were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics. They were stored frozen in our cell bank. All procedures were approved by the local Ethics Committee.
Preparation of a second generation of graftable bACLs.
The first generation of bACLs was produced using a scaffold anchored with two bone plugs (Goulet et al., 2000, 2004, 2006 , 2007 and 2011; Hart et al., 2005;
Tremblay et al., 2011). However, the second generation of bACLs wouldn’t contain
bone anchors anymore. The new bACLs would be fixed in situ using endobuttons CL BTB fixation device (Smith & Nephew). It provides strong, dependable ACL fixation and eliminates the need for knot tying, providing a strong and stiff repair. This also allows for more posterior placement of the femoral tunnel, if needed, and a 360° bone to graft contact.
Braided thread scaffold (sterile braided Vicryl Polyglactin 910 no 3, CCS-1, coated, Ethicon Inc, /Johnson & Johnson, Markham, ON, Canada, L3R 0T5), absorbable within 4-6 weeks in situ post-surgery, was passed through an endobutton. The thread itself is braided, but to further reinforce the bACL’s scaffold, three threads were braided together. The thread scaffold was transferred to a long sterile plastic tube and kept extended in a central, suspended position. The endobutton’s weight pulled the scaffold towards the bottom of the tube and kept is in place. Once the thread scaffold was ready, native Type I collagen was poured around it, filling the whole casting tube.
To cast the bACLs, DMEM containing FCS and 1.0 mg/ml of bovine type I collagen (solubilized in acetic acid diluted 1000 times with sterile water), was quickly mixed or not, with a suspension of goat DFs (2.5 x 105 cells in 1 ml). The mixture (total of 25 ml) was poured into a 30-ml sterile plastic tube, containing the surgical thread. The collagen polymerized within 20 min at room temperature, under a sterile culture flow hood while maintained without any agitation. Following polymerization, the collagen becomes a gel that must be covered with about 2 to 3 ml of DMEM supplemented with 10% FCS, 50 g/ml ascorbic acid and antibiotics. A cap was added to cover each tube. When the bACLs were seeded with cells, they were placed in an incubator at an atmosphere containing 8% CO2. They were maintained in DMEM supplemented with 10% FCS, 50 g/ml ascorbic acid and antibiotics. The acellular bACLs were also placed in the incubator, but in MEM without any additive.
After 24 hrs, all the bACLs were frozen in sterile Petri dishes overnight at -70°C and subsequently lyophilized. Then, they were transferred into new sterile plastic tubes. Following rehydration in fresh DMEM to produce a semi-rigid central core, a second coating of collagen seeded or not with DFs, could be performed as described above. Bilayered bACLs contained a lyophilized core. The bACLs were viable prior to implantation, since the cells progressively contracted the outer collagen layer in vitro over 24 hrs and thereafter. The bACLs containing DFs were sent for histological analyses, to see if the collagen fibers and the living cells were aligned in the same direction with the braided thread core. The acellular bACLs were used for surgical testing of the endobutton’s fixation technique.
Surgical testing.
The technical modifications of the bACLs implantation procedure were assessed in vitro on fresh bones of bovine knee joints. The approach of implantation using endobutton fixations has become widely used in orthopedic surgery. However, our bACL is a unique ACL substitute, produced entirely in vitro. It was important to
assess the feasibility of the bACLs implantation using endobutton devices. The use of a protective envelop during the passage of the graft in the bone tunnels had to be tested as well. Thus, 3 bACLs were implanted on fresh bovine knee bones in vitro, to assess the procedures.
Mechanical characterization of the braided thread scaffold.
Braided Vicryl threads (n=3) were subjected to uniaxial tensile testing on an Instron ElectroPuls E1000 mechanical tester. The stiffness (N /mm) and the ultimate tensile strength (Mpa) were measured during rupture assays performed on braided Vicryl scaffolds.
Histological analyses of the scaffold.
It was interesting to analyse the central braided thread core of the bACL after lyophilisation and rehydration, to assess the quality of the collagen matrix surrounding the braid. This work was performed by a private company, equipped to cut hard tissues such as surgical thread, in ultrathin slices of 10-15 um-thick.
RESULTS
Successful grafting of the first generation of acellular bACLs.
The first generation of bACLs was designed according to the bone-patellar tendon-
bone graft or BPTB graft, as it consists of tendon and bony attachments. Bones
were attached to the bACLs’ scaffolds. The grafting of such tissue-engineered grafts was performed the same way it is performed in humans. Figure 1 (A) shows one of the bACLs of the first generation that were grafted successfully in the goat model for 6 months. The bACLs were produced without cells, using native bovine Type I collagen as matrix. The advantage of adding living fibroblasts in the graft before implantation was the early initiation of caprine collagen synthesis and remodeling, slowly replacing the bovine collagen fibers. Thus, cell seeding is still suitable to produce autologous ACL substitutes. The bACLs were cultured under minimal tension and the collager fibers became aligned in a direction parallel to the tension applied (Fig. 1B). Endothelial cells colonized the bACLs in situ, as shown by the blood vessels (Fig. 1C; note the red or pink endothelial cells). The graft contained also nerve endings (Fig. 1D; brown structures and fibers). In addition, chondrocytes were observed at the interface between the ligament scaffold and each bone anchor, (Fig. 1E). After 6 months, the acellular graft was populated with cells, vascularised, innervated, and reinforced in situ (Fig. 1F), showing a macroscopic aspect that is very close to a native ACL (Fig. 1G), (13-15). Such bACL grafted in situ reached an average of 18% (94 + 14 N) of the native contralateral ACLs (520 + 59 N), (Table I).
Figure 1: (A): A bACL of the first generation grafted successfully in the goat model for 6
months. The bACLs were produced without cells, using native bovine Type I collagen as matrix. The bACLs were cultured under minimal tension and the collagen fibers became aligned in a direction parallel to the tension applied in absence (B) or in presence of cells (not shown). The bACL became vascularised (C: note the red or pink endothelial cells). The graft contained also nerve endings (D: brown structures and fibers). In addition, chondrocytes were observed at the interface between the ligament scaffold and each bone anchor, (E). After 6 months, the graft was remodeled, vascularised, innervated and reinforced in situ (F), showing a macroscopic aspect that is very close to a native ACL (G). (B-E, X40).
Table I: Mechanical features of acellular bACLs grafted for 6 months in the goat
model, compared to native ACLs used as controls.
Endobutton fixations of bACLs.
When anchored with bones, the diameter and the length of the bone plugs had to be relatively precise to respect the anatomic features of the knee joint. The length of the bACL also had to be measured. However, the length of the second generation of bACL can be variable, as it is pulled and then attached conveniently with the endobutton’s fixation. This makes the use of such graft adaptable to several knees. From its femoral attachment, the ACL has a length that ranges from 22 to 41 mm (mean, 32 mm) and its width from 7 to 12 mm (Amis and Dawkins, 1991).When a bACL is casted (Fig. 2A), the braided thread is placed in a sterile tube under the flow hood, and the weight of the endobutton pulls it at the bottom, while the solution of collagen is poured in the tube. After about 20 min, the collagen polymerises (Fig. 2B, 3A) and the bACL can be seeded or not with cells. If not, it can be readily frozen and lyophilized (Fig. 3B). Once rehydrated (Fig. 3C), an acellular bACL can be put in a cylindric plastic tube and stored at 4⁰C. The bACLs containing cells must be kept at 37⁰C, in a 8% CO2 incubator. The culture media must be changed every other day until use. Histological analyses of the scaffold revealed that the collagen adheres to the absorbable braided thread (Fig.
3D-E). Collagen fibers are aligned in the direction of the tension applied on the
tissue (Fig. 3D-E).
Tissue tested Stiffness (N /mm) Ultimate tensile strength (N) bACLs 64 + 11 94 + 14 ACLs 102 + 15 520 + 68
Figure 2: (A): A picture showing the casting step of a bACL of the second generation,