Reactive oxygen and nitrogen species induce protein and DNA modifications driving arthrofibrosis following total knee arthroplasty

A number of factors are known to result in complications after total knee arthroplasty (TKA), which include preoperative deformity, neuromuscular disease, patient noncompliance with rehabilitation protocol and technical errors such as component malpositioning [1–4]. A separate portion of the patient population develops arthrofibrosis after TKA, clinically defined as abnormal scarring of the joint in which the formation of dense fibrous tissue and tissue metaplasia prevent normal range of motion [4–11]. For these patients, surgical intervention and revision arthroplasty leads to a worsening of the fibrotic condition and eventual disability [4, 9, 10].

The exact pathoaetiology of arthrofibrosis following TKA remains elusive. However, aggressive fibroblast proliferation and tissue metaplasia are a hallmark of this condition [10, 12]. Our previous studies highlighted multiple tissue changes including the presence of pro-inflammatory factors, increased cell proliferation, survival and increased matrix deposition [11, 13]. In addition, we showed that mast cells, hypoxia and hypoxia-associated oxidative stress are linked to the progression of the metaplastic changes, fibrocartilage formation and heterotopic ossification observed in idiopathic arthrofibrosis [13].

Normally, tissue repair occurs through a sequence of coordinated events that lead to the eventual restoration of tissue form and function. The healing response is initiated by the clotting cascade, which results in the migration of inflammatory cells (neutrophils and monocytes) to the site of injury [14]. Inflammatory cell infiltration is followed by the recruitment of fibrocytes that undergo proliferation, differentiation and the ultimate deposition of an organized matrix [15–17]. Both the migration of inflammatory cells into the injured tissue and the proliferation of fibroblasts results in the release of cytokines, growth factors and reactive oxygen and nitrogen species (ROS/RNS) [18–26]. Thus, an intricate balance between cell proliferation, matrix production and tissue remodelling is in place during normal healing, and the restoration of tissue integrity is dependent on the coordinated or 'coupled' function of inflammatory cells responsible for remodelling, and fibroblasts, the cells responsible for resynthesis of the matrix. Once the process of healing nears completion, the majority of the inflammatory cells undergo apoptosis, the tissue heals and the release of ROS/RNS and other factors, which are no longer necessary, halts. Therefore, resolution of the inflammatory response is critical to the restoration of the tissue to a functional state and the prevention of fibrosis [16, 17].

Disruption of the ROS/RNS equilibrium, caused by overproduction or inefficient antioxidant response, has been implicated in the pathoaetiology of fibrotic conditions including retroperitoneal fibrosis [27], Dupuytren's [28–31], scleroderma [32, 33] and Crohn's disease [34]. In addition, chronic inflammation and oxidative stress contribute to genomic DNA damage. In Crohn's disease, this damage leads to the overexpression of p53, which potentially contributes to the loss of cell cycle control [35].

Our hypothesis was that susceptible patients exhibit excessive production and/or the inefficient removal of ROS/RNS after undergoing TKA surgery, which leads to aggressive fibrosis. Therefore, we performed a series of interlinked molecular studies to evaluate ROS/RNS modifications, inflammation and ROS/RNS responsive gene expression as part of the pathogenesis of arthrofibrosis following TKA.

This study highlights several major findings that are relevant to the pathogenesis of arthrofibrosis. First, histological findings show an increase in the number of macrophages and lymphocytes in the periarticular tissue of patients with arthrofibrosis. Second, by demonstrating the atypical presence of MPO and ROS/RNS products (oxidized DNA and nitrosylated proteins) in arthrofibrotic tissues, a potentially important mechanism involved in gene disregulation has been identified. Finally, by immunohistochemistry and microarray analysis, we show aberrant expression of MPO, SOD1 and other oxidative stress genes including; TrxR1, PNKP and MGST3, which all show significant fold changes in arthrofibrotic fibroblasts. Disregulated gene expression of the cellular oxidant/anti-oxidant system in fibroblasts implicates their involvement in the abnormal fibroblast proliferation, survival and hypertrophic formation of scar tissue.

No in depth studies have been performed on arthrofibrotic tissue after TKA in order to determine the extent of inflammation or other molecular mechanisms involved in this disease process [10, 11, 13]. However, chronic inflammation has been associated with the development of fibrosis in other tissues, such as Dupytren's Contracture [28, 30, 31, 42, 43], Crohn's disease [34] and additional tissues [14, 44]. Studies have also linked the development of arthrofibrosis after anterior cruciate ligament surgery to the presence of prior inflammation [45] and to an increased infiltration of T cells within 10 days after post surgery [46]. Similarly, in intestinal, pulmonary and renal fibrosis increased numbers of macrophages and mast cells are found within the granulation tissue during the proliferative stage of wound healing [20, 26, 27, 47]. Our findings, that macrophages and lymphocytes are present in the arthrofibrotic tissue, supports the involvement of chronic inflammation in the fibrotic process which develops after TKA. The persistence of this local inflammation, regardless of the number of years post initial surgery, was tightly linked to increased tissue calcification and decreased ROM. This association suggests that calcification and chronic inflammation are connected, which has been previously observed [48–52].

ROS/RNS is a collective term for a growing number of reactive species [37, 53, 54]. They are produced by a variety of pro-oxidant enzymes including MPO, which directly produces the highly reactive products hypochlorous acid (HOCl) and chlorine gas (Cl₂) [36, 55]. These products react with other ROS/RNS, leading to the generation of even a greater number of ROS/RNS [36–41, 53, 54]. In addition, HOCl and Cl₂ have been shown to mediate modifications of extracellular [56, 57] and intracellular components [40, 41]. Specifically, collagen is oxidized by a reaction with HOCl and Cl₂, resulting in the formation of chlorinated products. These modifications can affect the organization of the tissue matrix, altering its mechanical properties as well as preventing normal remodelling and resolution of the injury response. Intracellular modifications by MPO include ROS/RNS-mediated DNA hydroxylation and other base pair modifications, which affect gene expression.

In the present study, we show periarticular arthrofibrotic tissue with macrophages and fibroblasts uncharacteristically expressing high levels of MPO. The over-expression of MPO by macrophages and non-myeloid cells is also an aetiology associated with cystic fibrosis and hepatic fibrosis [58, 59]. The promiscuous expression in these fibrotic diseases is associated with a -463G/A polymorphism within the MPO promoter [58, 59]. Individuals can be born with this polymorphism or develop it as a result of ROS/RNS-mediated promoter mutagenesis [36–41, 58, 59]. Our findings suggest that MPO expression by fibroblasts may have a related aetiology and that MPO may be a driving force in the fibrotic process associated with arthrofibrosis.

ROS/RNS play diverse roles in wound healing - they regulate mast cell degranulation and the release of a number of enzymes, cytokines and growth factors (for example, transforming growth factor-β and connective tissue growth factor (CTGF)) that participate in normal wound healing and fibrosis [23]. They can also directly stimulate CTGF expression, fibroblast proliferation and matrix production [33, 37, 60]. In normal wound healing, the majority of inflammatory cells undergo apoptosis, the tissue heals and the release of ROS/RNS and other factors stop, thereby ending the cycle of proliferation and allowing for wound resolution [61]. In the arthrofibrotic tissue, however, we see the presence of inflammatory cells and ROS/RNS products years after the initial TKA.

We also observed a decreased expression of SOD1, which can exacerbate oxidative stress. An imbalanced and inefficient antioxidant response has been noted in other chronic inflammatory conditions. If the antioxidant response is not sufficient, as in Crohn's disease where an overall increase in ROS/RNS results because of an excessive accumulation of MPO in the tissue coupled with a decrease in hydroxyl radical scavengers, the balance is shifted [34]. As a result of the disturbed ROS/RNS equilibrium, the inflammatory and proliferative phases of wound healing do not resolve and an aggressive fibrotic response ensues.

Since ROS/RNS mediate downstream effects, a second confounding factor(s) must be involved in disease progression. The other important observation in our study was the increased hydroxylation of DNA, signifying DNA modification. Additional supportive evidence for DNA oxidative damage-induced modifications in arthrofibrotic fibroblasts was the 7.6-fold upregulation of PNKP, a key enzyme in the base excision repair pathway [62]. Specifically, PNKP is one of the primary proteins responsible for repair of oxidatively-induced DNA lesions and single strand breaks. In addition to the upregulation of PNKP, the expression of the oxidative stress responsive gene TrxR1 was increased 11.9-fold in arthrofibrotic fibroblasts. TrxR1 is an oxidoreductase enzyme that promotes cell growth, down-regulates the function of p53 induced apoptosis, regulates DNA synthesis and protects against oxidant damage [63]. Finally, there was 21.1-fold decrease in the expression of MGST3, an anti-oxidant enzyme that reduces lipid hydroperoxides and detoxifies lipid peroxidation end products such as 4-hydroxynonenal [64].

There was an increase in inflammatory cell numbers, MPO expression, ROS/RNS product accumulation and disregulation of ROS/RNS responsive gene expression. The overexpression of MPO and the presence of ROS/RNS products indicates that ROS/RNS production by inflammatory cells and fibroblasts within the arthrofibrotic tissue is excessive, due either to increased MPO or to the decreased cellular antioxidant expression of SOD1 and MGST3 or a combination of both. These observations strongly suggest an imbalance in the oxidant/anti-oxidant system regulating the normal resolution of the inflammatory and fibroblastic proliferative phases of wound healing. Based on these findings, we believe that aggressive periarticular fibrosis and unresolved healing in patients with arthrofibrosis results from an excessive accumulation of ROS/RNS, ROS/RNS-modified DNA and disregulation of oxidative stress responsive genes.