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DOI: 10.1016/J.JHSE.2007.05.017
The Inflammatory Response and Hyaluronan Synthases in the Rabbit Flexor Tendon and Tendon Sheath Following InjuryFrom the Department of Hand Surgery, Uppsala University Hospital, Uppsala, Sweden and the McCaig Centre for Joint Injury and Arthritis Research, University of Calgary, Calgary, Alberta, Canada Correspondence: Dr Monica Wiig, MD, PhD, Uppsala University Hospital, Uppsala, Sweden. E-mail:monica.wiig{at}akademiska.se
Using a rabbit model of flexor tendon injury, mRNA levels for a subset of relevant molecules involved in inflammatory and fibrotic processes were assessed by reverse transcriptase-polymerase chain reaction 3, 6, 12 and 24 days after injury. Increased levels of COX-2, IL-1β, MMP-13 and TIMP-1 mRNA were detected in both tendon and tendon sheath following injury, with each molecule exhibiting tissue and time-dependent changes. MMP-13 and TIMP-1 mRNA levels were markedly upregulated in both tissues, whereas COX-2 and IL-1β predominantly increased in tendon. Both hyaluronan synthase (HAS) 2 and 3 exhibited increases in mRNA levels in tendon tissue after injury, HAS 2 being more pronounced. These findings support the concept that healing in the flexor tendon and the sheath involve different molecular events and that each tissue may require unique modifications if healing is to be enhanced and adhesions reduced.
Key Words: flexor tendon injury • MRNA levels • hyaluronan synthases • interleukin-1β • cyclooxygenase-2 • inducible nitric oxide synthase Adhesion formation between tendons and the tendon sheath, resulting in a limited digital range of motion, is still a relatively common complication after flexor tendon repair. However, outcomes have improved significantly over recent decades with the development of new suture techniques and postoperative rehabilitation protocols. Further progress will probably demand direct modulation of adhesion formation by influencing the involved molecular processes (Silva et al., 2002). To optimise this approach will require additional understanding and knowledge of the healing processes in the tendons and the tendon sheath in order to identify suitable targets and to determine the best time to intervene. Flexor tendon healing occurs through both an intrinsic system of expansion of endogenous tenocytes and an extrinsic process of migration of exogenous cells (inflammatory cells, mesenchymal stem cells, and cells from the tendon sheath) into the injury site (Manske et al., 1984). The extrinsic mechanisms are believed to contribute to peritendinous adhesion formation and various approaches have been investigated to limit aspects of the extrinsic response and adhesion formation (Manske et al., 1984; Strickland, 2000). Factors involved in the extrinsic, inflammatory phase of flexor tendon healing are probably involved in adhesion formation. A number of molecules which regulate inflammation and fibrosis in other tissues have been studied (Dinarello, 2004; Shi et al., 2001; Wilgus et al., 2004), including hyaluronic acid (Hagberg, 1992; Moro-oka et al., 2000; Wiig and Abrahamsson, 2000).
In the present study, mRNA levels for matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1), the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), hyaluronan synthases 2 and 3 (HAS2 and HAS3), as well as the cytokines tumour necrosis factor- Therefore, the goal of this study was to assess the expression of these molecules in healing tendon and tendon sheath at 3, 6, 12 and 24 days after injury in order to determine whether the expression patterns in the two tissues differed during healing, and could be a possible target for manipulation in the future to improve healing outcomes.
Animal model of flexor tendon injury Thirty adult skeletally mature female New Zealand White rabbits weighing 3 kg (± 0.3 kg) were used in this study, with the approval of the Ethics Review Board. The rabbits were allowed unrestricted cage activity before and after surgery in standard size cages and were provided with a standard laboratory diet and water ad libitum. Prior to surgery, the animals were anaesthetised with an intramuscular injection of fentanyl-fluanisone (0.3 ml/kg body weight) (Hypnorm®, Janssen, Belgium) and midazolam (2 mg/kg body weight) (Dormicum®, Roche, Switzerland). Immediately before surgery, a single intravenous dose of 100 mg cefuroxime (Zinacef®, Glaxo, England) was administered to prevent infections. All surgical procedures were performed under sterile conditions in an animal operating facility, using a microscope and microsurgical instruments. All surgeries were performed by one of the authors (MW). After preparing the hind paws, the flexor tendons were divided above the ankle at the muscle/tendon junction to partially reduce the tensile load on the phalangeal sectiono the tendons, a procedure used by other authors in a rat model (Oshiro et al., 2003). The volar sides of the proximal phalanges of the third digits were incised unilaterally. After opening the flexor tendon sheaths with a longitudinal incision between the first and second pulleys, the superficial flexor tendons were resected in the area of the tendon sheath corresponding to zone II in humans. The deep flexor tendons were then divided completely with a sharp cut through the intermediate segment proximal to the distal joint (Berglund et al. 2004). A modified Kessler suture (5-0 Prolene, Ethicon®, Johnson and Johnson, Sollentuna, Sweden) was used as the core suture of the tendon, and a running suture (7-0 PDS, Ehicon®, Johnson and Johnson, Sollentuna, Sweden) as the circumferential suture of the tendon repair. A running 7-0 PDS suture was used to close the tendon sheaths. The hind paws were not immobilised or dressed. The rabbits were divided into five groups, with six rabbits in each group (n = 6 for each type of tissue sample at each time point). One group was not subjected to surgery and served as a set of non-operated controls. The rabbits were killed by an overdose of fentanyl-fluanisone (Hypnorm®). The flexor tendons and tendon sheaths which underwent surgery were harvested 3, 6, 12 and 24 days respectively after surgery in 8 mm segments (4 mm on each side of the suture). The segments were, rinsed in physiological saline and frozen in liquid nitrogen immediately after removal. There were no tendon ruptures in any of the groups.
RNA extraction
Reverse transcriptase-polymerase chain reaction
Statistical analysis The total RNA concentrations in the tendon and tendon sheath tissues in the control group, and at different time points after surgery, are presented as the mean and standard error of mean. The one-way ANOVA model of variance followed by Tukey’s post hoc test was used to make comparisons between specific mRNA levels in the different groups of tendons and tendon sheaths, respectively. Similarity of variance was verified using Levene’s test. Mean values and standard error of mean, for injured tissues at the different time points, are expressed as a percentage of mean of control values for the respective molecule. P values less than 0.05 were considered significant. The software used for the statistical analysis was STATISTICA, Statsoft, USA. Levels of mRNA for MMP-13 are presented as normalised integrated density values, since mRNA levels for this molecule were undetectable in the uninjured controls up to 40 PCR cycles.
Total RNA concentrations in normal and injured tissues In uninjured tendon tissue, the total RNA concentration was very low. The total RNA levels increased significantly after injury in both tendon and sheath, but both more rapidly and to higher levels in the sheath compared to the tendon (see Fig 1). Levels remained high in the tendon up to 24 days after injury, but had started to decline in the sheath by that time point.
Proinflammatory cytokine mRNA levels Levels of IL-1β mRNA were significantly elevated in tendon sheath and tendon tissue following injury, with levels 4 to 15 times as high as control values, respectively, at 3 days after surgery (Fig 2, panel A). The mRNA levels for this proinflammatory cytokine gradually declined subsequently and reached values not significantly different from control values by 6 and 12 days after injury, respectively.
In contrast to IL-1β, mRNA levels for another proinflammatory cytokine, TNF-  , were too low to be detected at all time points after injury and in the uninjured controls in both tendon and tendon sheath (data not shown).
COX-2 and iNOS mRNA levels Levels of iNOS mRNA could only be measured in injured tendon tissue at specific time points (3 and 6 days) after injury. Furthermore, mRNA levels for the inducible nitric oxide synthase in tendon sheaths were undetectable at all time points after injury (data not shown).
MMP-13 and TIMP mRNA levels
TIMP-1 mRNA levels significantly increased in both tendon and tendon sheath by 3 days after injury, reaching levels 2 to 3 times higher than control values, respectively (Fig 3, panel B). These increased levels then slowly declined in the tendon, reaching uninjured levels by 24 days post-injury. Levels tended to remain elevated in the sheath, but these were not significantly different from control values due to animal-to-animal variation at these later time points.
Hyaluron synthases (HAS2 and HAS3) mRNA levels
The present study identified a number of temporal and tissue-specific responses regarding changes at the mRNA level for a relevant subset of proinflammatory cytokines and enzymes, as well as hyaluronan synthases in the healing rabbit flexor tendon and tendon sheath. The results presented indicate that the cellular responses in these two tissues exhibit disparate temporal and qualitative patterns of gene expression, findings that may have significance when considering interventions to improve healing such as treatment with anti-inflammatory agents and modulation of hyaluronan expression. Prevention of adhesion formation after flexor tendon injury through anti-inflammatory therapies has shown some potential in animal models (Carstedt, 1987; Szabo and Younger, 1990). In the present study, it was found that COX-2 and IL-1β exhibited similar temporal alterations in the pattern of mRNA levels in the tendon tissues after surgery, and IL-1β has been reported to induce COX-2 mRNA expression in tendon (Tsuzaki et al., 2003). The response of tendons and the tendon sheaths to injury was evident early following injury, with maximum COX-2 mRNA levels in both tendon and tendon sheath occurring after 3 days and declining after the first week post-injury. However, COX-2 mRNA levels exhibited a trend towards sub-control levels at the later timepoints in both tissues. Thus, there is a possibility of reducing adhesion formation through inhibition of COX-2 during the first week following injury, but use after 1 week has not been supported by our findings. In this rabbit model, iNOS mRNA levels were undetectable in uninjured controls, but could be measured 3 days after surgery in tendon tissue, returning to undetectable levels of mRNA by 12 days post-injury. This pattern somewhat resembles the reaction to injury in extrasynovial Achilles tendon (Lin et al., 2001). In contrast, increases in MMP-13 (collagenase-3) mRNA levels in healing tendon peaked during the first 2 weeks post-injury and then started to decline by 24 days post-injury. Such a pattern may be consistent with the collagen degradation that is known to occur after injury (Oshiro et al., 2003). However, mRNA levels for this MMP in the tendon sheath remained at peak levels, even at the last time point assessed (24 days post-injury), a situation that could indicate that excessive matrix turnover persists in the healing sheath as the fibrosis and adhesions mature. Tendon sheath fibroblasts have been shown to respond to stimuli with a greater MMP production than endotenon fibroblasts (Khan et al., 1998), and this may be related to the inflammatory response associated with extrinsic healing and subsequent adhesion formation. The pattern of changes in MMP-13 mRNA levels following flexor tendon injury are similar to those detected previously in healing rabbit medial collateral ligament scar, where MMP-13 mRNA levels peaked 3 weeks after injury and then declined (Hellio Le Graverand et al., 2000). The major endogenous regulators of MMP activity are the tissue inhibitors of metalloproteinases (TIMPs), of which there are four types with the capacity to bind to all MMPs, but which differ in specificity (Brew et al., 2000). After injury, TIMP-1 mRNA expression in both tendon and sheath peaked at day 3 and then gradually decreased, exhibiting a similar pattern to that observed for MMP-13 in tendon. It is possible that this balance is disturbed in tendon healing with adhesion formation, but this awaits future confirmation.
To prevent adhesion formation and improve postoperative range of motion, an approach other than directly targeting the inflammation and matrix degradation in the healing tissue would be to modulate events at the tendon–sheath interface. One possible method to accomplish this goal is through modulation of hyaluronan (HA) expression/synthesis. HA has several important functions, partaking in the organisation of the extracellular matrix, angiogenesis, tumorigenesis and wound healing (Knudson and Knudson, 1993; Laurent and Fraser, 1992). Three HA synthases (HAS) have been identified in mammalian species (HAS 1-3) and these isoforms appear to be regulated differently and make HA of differing molecular weights (Ijuin et al., 2001; Itano and Kimata, 2002; Van den Boom et al., 2006). Both HAS1 and HAS2 produce high molecular weight HA, while HAS3 produces HA of low molecular weight. Thus, the products of the isoforms may serve different functions in different situations. IL-1β and the two hyaluronan synthases assessed (HAS2 and -3) exhibited similar patterns of alterations in mRNA expression in tendon and tendon sheath following injury, findings that support an interrelationship. It has been shown in other types of tissue that both IL-1β and TNF- The present study would also indicate that the regulation of expression of this synthase is very different in the tendon and the sheath following injury, and, thus, up-regulation in the sheath may be a critical step on which to focus future efforts. Although the duration of the present study did not include the later remodelling phase of healing/repair that follows, the events initiating adhesion formation are likely to occur early during the inflammatory and proliferative phases of the healing process. However, in future studies targeting adhesion formation, it will be of interest to include later time points and biomechanical parameters to ascertain that the treatment does not reduce ultimate tendon strength and increase the risk of tendon rupture. A possible limitation of the present study is that mRNA expression may not completely mirror the protein levels, since there is post-transcriptional regulation of protein synthesis. Nevertheless, our findings are consistent with those reported by other investigators, and previous studies measuring mRNA and protein levels have indicated a good correlation (Boykiw et al., 1998; Wang et al., 2002, 2003). In a rabbit model, the clinical situation with passive or restricted active rehabilitation after flexor tendon injury is not practically possible to design. Instead, the partial unloading of the tendon, via transection of the tendon at the muscle/tendon level, reduces the force and risk of ruptures while leaving enough tension for tendon excursion with toe movement. In summary, it has been demonstrated that mRNA levels for a number of molecules implicated as stimulators or inhibitors of fibrosis and scarring are up-regulated during the first phases of flexor tendon healing and that there are some differences between tendon and sheath tissue, indicating tissue-specific responses to injury and requirements for optimal healing (see Table 2). It has been shown that the increases in COX-2 are high in tendon during the initial inflammatory phase of healing but then decline, even subsequently tending towards suppression in the sheath, implying that any treatment targeting COX-2 would only be relevant during the first week after injury. Other targets for future investigations include HAS2 and iNOS, where stimulating production of hyaluronan and nitric oxide could inhibit both inflammation and adhesion formation (Murrell et al., 1997).
The authors thank Carol Reno for excellent technical assistance in their studies. These studies were supported by funds from the Centre for Research and Development Gävleborg – Uppsala University, K. and A. Wallenbergs Foundation, G. and J. Aners Foundation (Sweden) and the CIHR Institute for Gender and Health (Canada). Dr DA Hart is the Calgary Foundation-Grace Glaum Professor in Arthritis Research at the University of Calgary. Received for publication June 26, 2006. Accepted for publication May 30, 2007.
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Abstract
MATERIALS AND METHODS
. Archives of Oral Biology, 46: 767–772.