Enhancement of tendon-bone interface healing and graft maturation with cylindrical titanium-web (TW) in a miniature swine anterior cruciate ligament reconstruction model: histological and collagen-based analysis

Ryu et al. BMC Musculoskeletal Disorders (2020) 21:198 https://doi.org/10.1186/s12891-020-03199-0 RESEARCH ARTICLE Open Access Enhancement of tendon-bone interface healing and graft maturation with cylindrical titanium-web (TW) in a miniature swine anterior cruciate ligament reconstruction model: histological and collagen-based analysis Keisho Ryu*, Mitsuru Saito, Daisaburo Kurosaka, Seiichiro Kitasato, Toshiyuki Omori, Hiroteru Hayashi, Tomohiro Kayama and Keishi Marumo Abstract Background: Tendon-bone interface healing and ligamentization of the graft in anterior cruciate ligament (ACL) reconstruction with autografts are important factors affecting treatment outcome. This study aimed to investigate the effectiveness of a cylindrical titanium-web (TW) in tendon-bone interface healing and graft maturation in ACL reconstruction. Methods: Fourteen mature female CLAWN miniature swine underwent bilateral ACL reconstructions with patellar tendon (PT) autografts. In one limb, the TW/tendon complex was placed into the proximal side of the tibial tunnel. Only the graft was transplanted into the tunnel in the control limb. The proximal side of the graft was sutured into the stump of the native ACL and the distal end was stapled to the tibia. The animals were euthanized at 4 and 15 weeks postoperatively, for histological and biochemical analyses. Results: Microscopic images in TW limbs showed that ingrowth of tendon-like tissue and mineralized bone tissue into the TW connected the bone and the tendon directly. In contrast, fibrous tissue intervened between the bone and tendon in the control limbs. The total amount of collagen cross-links (which defines the strength of collagen fibers) and the maturation of collagen cross-links in TW tendons were significantly higher (p < 0.05) than those of control limbs. There was no significant difference in the ratio of dihydroxy-lysinonorleucine to hydroxylysinonorleucine (an indicator of tissue specific collagen maturation) between TW tendons and that of the native PT. Conclusions: TW promoted the maturation and formation of collagen cross-links in the grafted tendon while maintaining the cross-links pattern of native tendon collagen, and enabled direct binding of tendon to bone. Keywords: ACL reconstruction, Enthesis, Tendon-bone interface, Titanium-web, Miniature swine, Collagen maturation, Ligamentization * Correspondence: ; Department of Orthopaedic Surgery, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Tokyo, Minato-ku 105-8461, Japan © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ryu et al. BMC Musculoskeletal Disorders (2020) 21:198 Background Anterior cruciate ligament (ACL) injury is one of the most common knee injuries in teenagers and young adults. Approximately 250,000 individuals, in the USA alone, suffer from ACL injury per year [1]. Usually ACL reconstruction with autografts or allografts is performed to restore function after the injury, and around 175,000 ACL reconstruction surgeries were performed in the USA annually [2]. However, delayed sports recovery and risk of re-rupture remain problematic, and many researches attempt to recreate the native ACL. Tendonbone interface healing and ligamentization of the graft in ACL reconstruction with autografts are important factors affecting treatment outcome after surgery [3, 4]. After ACL reconstruction with a free-tendon graft, collagen fibers are first formed at the tendon-bone interface, and then collagen fibers perpendicular to the interface called the Sharpey-like fibers form to connect the tendon and bone tunnel [5, 6] . This so-called indirect insertion is different from the direct insertion found in the normal ACL tendon–bone interface or tendon–bone late healing phase at the exit of the bone tunnel [5, 7]. Direct insertions are characterized by connection of the graft and bone tunnel through the fibrocartilage tissue, forming the “tidal line” structure that stains positively for basophilia [5, 7]. Most researchers believe that indirect insertions are less effective than direct insertions and this slow and incomplete healing of the tendon-bone interface may result in inferior functional recovery and even worse osteoarthritic changes [8]. The transplanted tendon into the knee joint during ACL reconstruction undergoes a biological transformation process with changes in vasculature and collagen profiles. This remodeling process into a viable ACL-like tissue is termed “ligamentization” [4, 9]. The time required for ligamentization after ACL reconstruction has been reported to range from 9 to 18 months under light microscopy [4], 13 to 30 months under electron microscopy [4], and within 1 year in a collagen cross-links analysis [10]. The current challenge is to find a better strategy to improve tendon-bone interface healing and promote maturation of the grafted tendon for faster recovery. Mesenchymal stem cell transplantation [11–14] and administration of biological growth factors, such as recombinant human bone morphogenetic protein-2 (rhBMP-2) [6, 14, 15], transforming growth factor (TGF) -beta1 [16], fibroblast growth factor (FGF) [5] and granulocyte colony stimulating factor (G-CSF) [17], have been performed in recent studies. Though most studies using growth factors failed to form fibrocartilage tissue at the tendon–bone interface, Hashimoto et al. reported that they succeeded in generating a tendon-bone junction similar to normal enthesis using rhBMP-2 in a Page 2 of 12 rabbit model [15]. However, this method included injecting rhBMP-2 into the flexor digitorum communis tendon to induce ectopic ossicle formation, which was then surgically transferred into the tibia [15]. This method has problems in clinical application, such as the biological effects caused by growth factors and newly recruited mesenchymal stem cells, as well as high cost, and cumbersome surgical procedure. Strong, biodegradable materials are ideal material (...truncated)