Agosto2012;38(suppl.2):26-30s26

A. sPEZIALI* **, K.F. FARRARo*, K.E. KIM*, s.L-Y. Woo* * Musculoskeletal Research Center, Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, 405 Center for Bioengineering, Pittsburgh, PA, USA; ** Department of Orthopaedics and Traumatology, University of Perugia, Italy

Indirizzo per la corrispondenza:Savio L-Y WooMusculoskeletal Research Center, Department of Bioengineering, Swanson School of EngineeringUniversity of Pittsburgh, 405 Center for Bioengineering, 300 Technology Drive, Pittsburgh, PA 15219, USATel. +1 412-648-2000 - Fax +1 412-648-2001E-mail: ddcenzo@pitt.edu

RiassuntoBackground. I progressi nell’ingegneria tessutale hanno miglio-rato la guarigione dei tessuti connettivi molli attraverso lo svilup-po di sommazioni biologiche e meccaniche. obiettivi. Fornire un’esauriente review di studi di laboratorio, esplorando come queste sommazioni possano implementare la guarigione di tendini e legamenti. Metodi. La sommazione biologica, usando bioscaffolds di matri-ce extracellulare, fu utilizzata per trattare un difetto patellare, un gap del LCM ed un LCA sezionato. Successivamente, la som-mazione meccanica, usando una sutura di addizione, fu usata per assistere la guarigione del LCA. Gli incoraggianti risultati condussero all’uso combinato della sommazione biologica e meccanica. Infine, un nuovo scaffold metallico biodegradabile fu introdotto per la guarigione del LCA .Risultati. A 12 settimane , la sommazione biologica aumentò la rigidezza del difetto patellare del 98% ed incrementò il modulo tangente del LCM del 33%. Per il LCA, la sommazione biologica aumentò la rigidezza del femur-ACL-tibia-complex del 140%; la sommazione meccanica ridusse la translazione tibiale antero-posteriore del 20% rispetto al gruppo della sutura di riparazione a 12 settimane. Conclusioni. La sommazione biologica e meccanica ha dimon-strato di migliorare la guarigione di tendini e legamenti. Per il LCA, rimane necessario lavorare sulla protezione del neo-tessuto, così come sul tensionamento dei siti d’inserzione per prevenire la loro atrofia da disuso.Parole chiave: Guarigione legamento crociato anteriore, som-mazione biologica, sommazione meccanica, bioscaffolds matri-ce extracellulare, materiali metallici biodegradabili

HEALINg oF LIgAMENts AND tENDoNsThe healing of ligaments and tendons is generally slow. Laboratory studies have helped to understand that during the early phase of healing, the injured ligament needs protection from further damage, and later it needs ad-equate loading to promote and accelerate the remodeling of the healed tissue 1. Also, the healing process of injured ligaments and tendons can vary widely and therefore, the management of a torn anterior cruciate ligament (ACL) 1-3

is not the same as that of a torn medial collateral ligament (MCL). In our research center, we have used a canine model to show that a transected MCL could heal spontaneously without surgical repair nor immobilization  1. The varus-valgus (V-V) knee rotation was restored to control levels by 12 weeks with the structural properties of the femur – MCL – tibia complex (FMTC) were completely recovered to the level of sham - operated controls. These findings coincided with satisfactory clinical outcome following functional treatment of an isolated grade III MCL tear 4 5. However in a more realistic “mop-end” tear model in the rabbit 2 the V-V knee rotation recovered, and the stiffness of the FMTC could recover by 12 weeks, but the entire healing process of the ligament substance and its inser-tion sites were not complete even after one year 6. The

BIoLogICAL AND MECHANICAL AUgMENtAtIoN FoR HEALINg oF LIgAMENts AND tENDoNssommazioni biologiche e meccaniche per la guarigione dei legamenti e tendini

summaryBackground. Advances in tissue engineering have enhanced the healing of soft connective tissues through biological and mecha-nical augmentation. objectives. To provide a comprehensive review of laboratory studies, exploring how these augmentations could improve the qualities of healing tendons and ligaments. Methods. Biological augmentation, using an extracellular matrix bioscaffold, was used to treat a patellar defect, a MCL gap inju-ry, and a transected ACL. Subsequently, mechanical augmenta-tion, using a suture augmentation, was used to assist a healing ACL. The positive findings led to a combined use of biological and mechanical augmentation. Finally, a novel biodegradable metallic scaffold for ACL healing was introduced. Results. By 12 weeks, biological augmentation increased the stiffness of a patellar defect by 98% and improved the tangent modulus of a healing MCL by 33%. For the ACL, biological augmentation increased the stiffness of the healing femur-ACL-tibia complex by 140% and mechanical augmentation reduced anterior-posterior tibial translation to 20% of the suture repair group at 12 weeks.Conclusions. Biological and mechanical augmentation has been shown to improve the healing of ligaments and tendons. For the ACL, much work remains including protection of the neo-tissue during healing as well as loading of its insertion sites to prevent disuse atrophy.Key words: anterior cruciate ligament healing, extracellular ma-trix bioscaffolds, biological augmentation, mechanical augmen-tation, biodegradable metallic materials

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mechanical properties of the healing MCL (tissue quality) remained poor, with concomitant elevation of its collagen V/I ratio. It was concluded that the improved function of the FMTC was a result of increase in the cross sectional area (tissue quantity).In recent years, the goat model has become the pre-ferred animal model for studies of healing of knee liga-ments and tendons due to its robust activity level and a larger stifle joint. Using the “mop-end” tear model in goats the healing MCLs were assessed at 12 weeks  3. Gross examination of the healed MCL showed enlarged neo-tissue adhered at the site of the “mop-end” tear, with a cross sectional area (CSA) almost twice that of the sham-operated control. Using the novel robotic / universal force - moment sen-sor (UFS) testing system, developed in our laboratory  7 (Fig. 1), it was found that the injured MCL had 40% high-er V-V knee rotation than that of the sham-operated control at all flexion angles.

In spite of these differences, the stiffness, obtained from the tensile testing of the FMTC, was 61.8 ± 19.6 N/mm, and was not significantly different from that of the control (72.7 ± 9.2 N/mm, p < 0.05). However, its ultimate load was approximately 60% that for the control (450 ± 146 N vs 765 ± 149 N, p < 0.05). From these studies, it was concluded that an isolated MCL tear can heal with func-tional treatment, within weeks, but the remodeling process will take months. Unlike the case of isolated MCL injuries, the management of combined injuries of the ACL and MCL is still controver-sial, because there are excessive loads on the MCL due to the loss of the ACL would impair its healing. Combined

ACL and MCL injury was studied in our research center using dog, rabbit, and goat models. It was found that complete loss of the ACL resulted in poor healing of the MCL at 6 and 12 weeks after injury 8 9. Additionally, V-V knee rotation of the stifle joints also failed to return to normal levels. These studies clearly indicated that there is a need to find ways to improve MCL healing. With the advent of tissue engineering (TE), using cells, bioactive molecules (growth factors and cytokines), and scaffolds, the potential to im-prove the properties of healing tissues has become a real-ity. In our research center, both biological and mechanical augmentation techniques were explored to improve the histological, biochemical, and biomechanical properties of the healing patellar tendon (PT), MCL, and ACL.

BIoLogICAL AUgMENtAtIoN

the porcine small intestine submucosa: a “smart” bioscaffoldRecently, our research center has used the porcine small intestine submucosa (SIS), an extracellular matrix bioscaf-fold (ECM-SIS), to promote the healing of ligaments and tendons 10-12. ECM-SIS has aligned collagen type I fibers that could guide the neo-tissue formation, as well as provide growth factors, such as FGF, TGF-β, VEGF, and fibronectin 13, that would result in chemotaxis of inflammatory cells, and fi-broblasts to accelerate neo-tissue production. Patellar Tendon Healing. Donor site morbidity has been one of the main concerns following PT graft harvesting for ACL reconstruction. The consequences could be ante-rior knee pain 14 and osteoarthritic changes 15. It was hy-pothesized that ECM-SIS could enhance cell proliferation, matrix production, and biomechanical properties of the neo-PT as well as limit the cellular and vascular infiltration from the fat pad; thus, reducing donor site morbidity. In a rabbit model, two layers of ECM-SIS were applied to a central third PT defect 10. The first layer was placed posterior to the PT defect, with the abluminal side facing the defect, while a second layer was placed anteriorly, again with its abluminal side toward the defect to facili-tate healing. At 12 weeks, the SIS-treated PT defect achieved a 68% increase in CSA, 98% increase in stiffness, and 113% increase in ultimate load in comparison to the non-treated group. In addition there were no noticeable adhesions between the neo-PT and the fat pad. These findings suggest that the use of ECM-SIS could im-prove the structural properties of the healing PT by accel-erating tissue formation, and prevent the formation of ad-hesions; a process that could reduce donor site morbidity. Medial Collateral Ligament Healing. A single sheet of ECM-SIS applied to a 6 mm MCL gap injury was also

FIg. 1. Goat stifle joint mounted on the robotic / UFS testing system. With permission of Woo 1998 7.

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found to improve the mechanical properties of the FMTC after 12 and 26 weeks of healing in a rabbit model 11. Gross inspection of the healed MCL at 26 weeks revealed a continuous neo-tissue across the gap, and the CSA was closer to the sham-operated controls compared to the non-treated group. Histologically, patches of larger collagen fibrils in addition to a lower collagen type V to I ratio were found in the SIS-treated group. Concomitantly, the tangent modulus was 33% higher in the SIS-treated group than that for non-treated group (387 ± 99 MPa vs 291 ± 123 MPa, p < 0.05). The tensile strength of the SIS-treated group (36 ± 11 MPa) was 50% higher than the non-treated group (24  ±  9 MPa), indi-cating improved tissue quality. However, these properties were still lower than the sham-operated control (84 ± 19 MPa, p < 0.05). These findings had revealed that ECM-SIS could guide the healing process by inducing better organization of colla-gen fibers, limiting tissue hypertrophy, that resulted in an improvement of the quality of the neo-ligament.

Anterior cruciate ligament healingIt is well known that a tear of the ACL, especially at its midsubstance, has a very low healing capacity. For this reason, the gold standard for clinical treatment has been reconstruction using an autografts or allografts. Unfortu-nately, long term follow-up studies have shown that many patients would end up with symptoms of osteoarthritis on their knees 16. With TE, a renewed effort has been made to heal a torn ACL. Steadman et al. have treated proximal ACL tears by creating small microfractures around the femoral footprint of the ACL to promote “healing response” 17. In a labo-ratory study, Joshi et al. 18 found that a collagen-platelet composite (CPC) scaffold in addition to a primary suture repair could stimulate the healing of a midsubstance transection of the porcine ACL. In our research center, a combination of ECM-SIS sheet and hydrogel was used to treat a transected ACL in the goat model 12. To address the concerns of acute or chronic immune rejection, the ECM-SIS and hydrogel were ob-tained from genetically modified α-Gal epitope-deficient pigs 19. The two ends of a transected ACL were first repaired by a suture repair (SR) technique (Fig.  2/a). The ECM-SIS sheet was wrapped around the ACL stumps and sutured in place (Fig. 2/b). Then, the SIS hydrogel was injected into the injury site (Fig. 2/c) 20. The hypotheses understudy were that the combined use would provide an abundant source of growth factors to accelerate ACL healing, as well as limited neo-tissue hypertrophy. After 12 weeks, the SIS-treated ACL healed with continu-ous neo-tissue without signs of hypertrophy (Fig. 3). The

CSA (29.0 ± 19.3 mm2) was comparable to that of the intact ACL (23.0 ± 4.6 mm2) and, it was 4.5 times larger than the SR alone group (6.5 ± 4.3 mm2). Histologically, SIS-treated tissue samples had collagen fib-ers parallel to the longitudinal axis of the ligament with many spindle-shaped cells.Functionally, the anterior-posterior tibial translation (A-PTT), as measured by the robotic / UFS testing system, for both the SIS-treated and the SR-treated groups were higher compared to intact ACLs (p < 0.05). But, the in situ force of the SIS-treated ACLs was not statistically different from their sham-operated controls (p < 0.05), and it was higher than the SR alone group (52 ± 8 N vs 29 ± 25 N, at 60° of flexion).Tensile testing of the femur – ACL – tibia complex (FATC) showed the stiffness for SIS-treated group was 140% high-er than that the SR group (53 ± 19 N/mm vs 24 ± 21 N/mm, p < 0.05). It should be noted, however, these proper-

FIg. 2. Biological augmentation for the ACL: (a) suture repair (SR); (b) wrapping of ECM-SIS sheet; (c) SIS-hydrogel injection. With permission of Fisher, Woo 2011 12.

FIg. 3. Visual inspection of the ACLs at 12 weeks of healing in the goat model: (i) sham-operated ACL; (ii) SIS-treated healing ACL; (iii) suture repaired (SR) healing ACL. With permission of Fisher, Woo 2011 12.

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ties were significantly lower than sham-operated FATCs (p < 0.05).The results of this study showed that ACL healing is possi-ble, nevertheless the process of healing is slow. Thus, it is necessary to find ways to restore initial joint stability, such as mechanical augmentation to avoid further damage to other soft tissues around the knee.

Mechanical augmentation for ACL healingSuture augmentation (SA) was found to be able to restore initial joint stability in the goat model  21. When sutures were passed directly from bone to bone and tied under tension, using a large knot on the femoral side and a sur-gical button on the tibial side (Fig. 4/a), it could restore the A-PTT within 3 mm of the intact joint. When compared with SR alone (Fig.  4/b) the A-PTT remained 5-6 times higher than the normal value (p < 0.05).

In Vivo animal study. After 12 weeks of healing in a goat model, the gross morphology of the SA group revealed continuous neo-tissue formation, while the augmentation sutures were loose. The CSA of the SA group was closer to that of the sham-operated controls, and also was 2.3 times higher than that of the SR alone group.The SA group also had improved joint stability. The A-PTT was 21%, 21%, and 14% lower than those of the SR group at 30°, 60° and 90° of flexion, respectively, but all were still significantly higher than those for the sham-operated controls (p < 0.05). The in situ force for the SA-treated ACLs was also 54%, 60%, and 131%, respec-tively, higher than SR-treated ACLs (p < 0.05). Tensile testing of the FATC showed that the stiffness for the SA group was 75 % higher than that for the SR alone group (52 ± 21 N/mm vs 24 ± 21 N/mm, p < 0.05). Again, it was below the levels of their sham-operated con-trols (152 ± 29 N/mm, p < 0.05).

This series of studies demonstrated that both biological and mechanical augmentation could enhance ACL healing. Still, neither was able to completely restore a transected ACL back to the native ACL. Thus, an effort by combining bio-logical and mechanical augmentation is underway to exam-ine their synergistic effects in accelerating the ACL healing.

oN-goINg stUDIEs

Biodegradable metallic materials for mechanical augmentationA preliminary study also found that between 12 to 26 weeks of healing, tensile testing of the SIS-treated goat FATC had changed its tensile testing of failure mode from the midsubstance to the insertion sites. This finding suggest disuse atrophy of the insertion sites during the lengthy heal-ing process 12. Thus, loading a torn ACL during the early healing process would be needed. As such, we are explor-ing new ways to add mechanical loading to the torn ACL, and thus its insertion sites, to limit this detrimental effect. Based on an in-vitro study using a biodegradable magne-sium (Mg) - based ring, sutured around the circumference of the two stumps of a surgically transected ACL in the goat stifle 22 (Fig. 5), it was found that the stiffness of the FATC could reach 22.5  ±  4.5 N/mm, which was statistically higher than those for the SA alone (16.0 ± 1.5 N/mm, p < 0,05). These early results are encouraging as they suggest that the ACL, and therefore its insertion sites are loaded with the Mg-based ring in place. Future in vivo studies are planned to examine whether this initial loading translate to maintaining the integrity of the ACL insertion sites. In the end, the use of Mg-based ring together with SA, ECM-SIS sheet, and hydrogel may be the biological and mechanical augmentation that would work in synergy in healing of a torn ACL, by providing initial joint stability and loading of the ACL.

FIg. 5. Schematic model for the mechanical augmentation of the ACL: combined Mg-based ring and suture augmentation (SA). With permission of Farraro, Woo 2012 22.

FIg. 4. Mechanical augmentation for the ACL: (a) suture augmentation (SA), and (b) suture repair (SR) in a goat model. With permission of Fisher, Woo 2011 21.

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