Timing of administration of transforming growth factor-beta and epidermal growth factor influences the effect on material properties of the in situ frozen-thawed anterior cruciate ligament
Introduction
It has been well known that the material properties of a tendon autograft are reduced after ligament reconstruction surgery, and that they remain abnormal even at 12 months after surgery (Amiel et al., 1986; Arnoczky et al., 1982; Butler et al., 1989; Chimich et al., 1992). These changes in the autograft are induced partly by extrinsic fibroblast proliferation and ingrowth along with revascularization, which occur after intrinsic fibroblast necrosis (Kleiner et al., 1989). Therefore, one of the future goals in ligament reconstruction is to prevent graft deterioration after transplantation or to accelerate mechanical restoration of the deteriorated graft. Recently, several studies have reported in vivo effects of growth factors on healing of the injured medial collateral ligament (Batten et al., 1996; Hildebrand et al., 1998; Letson and Dahners, 1994; Weiss et al., 1995; Woo et al., 1996). Woo et al. (1996) reported that a combined administration of 4-ng TGF-beta and 100-ng EGF to the injured medial collateral ligament significantly decreases the cross-sectional area and significantly increases the tangent modulus at 6 weeks after injury. Therefore, there is a high possibility that these growth factors may prevent the graft deterioration after transplantation. No studies, however, have been conducted to clarify in vivo effects of growth factors on the tendon graft or its model.
The in situ frozen-thawed ligament and tendon tissues have been established as an idealized model of anatomic but acellular autograft (Jackson et al., 1991; Katsuragi et al., 2000; Kleiner et al., 1989; Majima et al., 1994; Ohno et al (1993), Ohno et al (1996); Sakai et al., 1999; Tohyama and Yasuda, 2000; Tsuchida et al., 1997; Yasuda and Hayashi, 1996). Our previous study (Katsuragi et al., 2000; Sakai et al., 1999) showed that intrinsic cells in the frozen-thawed tendon and ligament tissues are necrotized after the treatment, and the tissues are thoroughly acellular for 2 weeks. In the acellular phase, no significant changes are observed in the material properties, the cross-sectional area, and the water content. Extrinsic fibroblasts start to infiltrate into the surface portion of the frozen-thawed ACL at 3 weeks after treatment. The surface portion of the ACL has been already filled with numerous fibroblasts at 6 weeks, although the core portion remains acellular. It takes a few months for the acellular area in the core portion to disappear. In the cell proliferation phase, the cross-sectional area of the frozen-thawed tendon significantly increases and the material properties significantly decrease over time (Jackson et al., 1991; Majima et al., 1994; Ohno et al (1993), Ohno et al (1996)). Kleiner et al. (1989) and Tohyama and Yasuda (2000) showed that extrinsic cell proliferation and revascularization cause the deterioration not only in the free tendon autograft but also in the frozen-thawed tissues. Ultrastructurally, Jackson et al. (1991) and Tsuchida et al. (1997) reported that the population of small-diameter fibrils increases in the frozen-thawed tissues between 6 weeks and 6 months, suggesting that formation of new small-diameter fibrils has occurred. Thus, it has been recognized that the in situ frozen-thawed tendon or ligament model has a similarity to the free tendon autograft model.
In the present study, therefore, we hypothesized that 4-ng TGF-beta1 and 100-ng EGF applied around the ACL at 3 weeks after the freeze-thaw treatment may significantly affect its material properties, and that the timing of administration, which is performed at the 3- or 6-week period, may influence the effect of TGF-beta and EGF on the frozen-thawed ACL, because these growth factors can affect cells via receptors on the cell surface. The purpose of the present study is to test these hypotheses.
Section snippets
Study design
This multidisciplinary study was performed with 68 mature female Japanese White rabbits weighing 3.5±0.3 kg (mean and standard deviation). Animal experiments were carried out in the Institute of Animal Experimentation, Hokkaido University School of Medicine under the Rules and Regulation of the Animal Care and Use Committee, Hokkaido University School of Medicine. In each rabbit, we applied the in situ freeze-thaw treatment to the right ACL using the below-described surgical technique. After
Tissue dimension and material properties of the ACL substance
The ANOVA demonstrated a significant difference (p=0.0004) in the cross-sectional area among the 5 groups (Table 1). Group G/F3 was significantly lesser than Group F3 (p=0.0062), while there were no significant differences between Groups G/F6 and F6. Group G/F3 was also significantly lesser than Group G/F6 (p=0.0268), while there were no significant differences between Groups F3 and F6. There were no significant differences between Group G/F3 and the control data, while Groups F3, G/F6 and F6
Discussion
The present study demonstrated that 4-ng TGF-beta and 100-ng EGF applied at 3 weeks after the freeze-thaw treatment significantly inhibited several effects of the freeze-thaw treatment on the ACL at 12 weeks. Specifically, it significantly inhibited the increase of the water content and the cross-sectional area due to the freeze-thaw treatment with the vehicle, and inhibited the material deterioration of the ACL. However, the same dose of the growth factors applied at 6 weeks did not show any
Acknowledgments
This work was supported financially in part by the Grant-in-Aid for Scientific Research (Nos. 12470299 and 12671389), from the Ministry of Education, Science and Culture, Japan. This study was presented in part in the 47th Annual Meeting of Orthopaedic Research Society, San Francisco, USA, in 2001.
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