Elbow Stiffness: Basic Science and Overview

Cytokines

A complex system of chemical and mechanical signals modulates the epithelial to mesenchymal transition and the subsequent differentiation of mesenchymal stem cells (MSCs) and fibroblasts into myofibroblasts, as well as the activity of the latter (see Fig. 53.2 ).

Chemical promoters of myofibroblast activity include transforming growth factor beta (TGF-β1), connective tissue growth factor, and the domain of fibronectin ED-A, which are elevated in posttraumatic joint capsules of both human elbows and animal stiffness models. TGF-β1 promotes connective tissue fibrosis by stimulating fibroblast migration and proliferation, myofibroblast differentiation, and collagen synthesis. Myofibroblasts may themselves produce and stimulate TGF-β1 activity, hence leading to a self-propagating TGF-β1–mediated activation. Myofibroblast contracture can activate latent TGF-β1 providing an interlink between chemical and mechanical signals.

Downregulators of myofibroblast activity have also been described. Tumor necrosis factor alpha (TNF-α) was shown to promote myofibroblast proliferation at low doses but inhibit matrix contraction at higher doses. This regulation was via the inhibition of α-SMA and collagen 1 gene expression. It was mediated through prostaglandin E2 and inhibited by diclofenac.

Mast Cells

Another mechanism contributing to myofibroblast activation is the mast cell–neuropeptide fibrosis axis. The mast cell–neuropeptide fibrosis axis is well documented in healing skin. Mast cells are found in joint capsules and contain granules of profibrotic mediators, including platelet growth factor A, endothelin 1, basic fibroblast growth factor, and TGF-β1. These profibrotic mediators can induce myofibroblast differentiation and proliferation. Substance P (SP) and calcitonin G are neuropeptides released from nerve terminals in response to injury and pain that stimulate mast cell degranulation.

In contracted capsules of both animal and human elbows with posttraumatic stiffness, the proportion of myofibroblasts, mast cells, and neuropeptide-containing nerve fibers is greater than compared to that of normal capsules. These changes are seen in acute and also chronic stiffness. In a rabbit model, mRNA levels of TGF-β1 and α-SMA were significantly increased in deep flexor tendons after injury and repair. Myofibroblasts, mast cells, and neuropeptide-containing nerve fibers were also significantly increased in healing tendons, supporting the involvement of the profibrotic neuropeptide–mast cell–myofibroblast pathway in deep flexor tendon healing. Hildebrand et al. isolated joint capsule cells from human elbow capsules, mixed them with collagen gels, and assessed the size of the resultant gel contraction. The joint capsule cells contracted collagen gels in a dose-dependent manner. This collagen gel contraction was enhanced by the addition of mast cells and increased further by the addition of SP. The SP receptor antagonist, RP67580, and the mast cell stabilizer, ketotifen fumarate, decreased the magnitude of contraction.

Although these findings provided a direct link between the myofibroblast–mast cell–neuropeptide axis and mechanical tissue contracture, unfortunately, our lab did not find a clinically reduced joint contracture after inhibition of SP with fosaprepitant, which competitively binds to neurokinin 1, the receptor for SP, in a rabbit model of joint contracture. Interestingly, however, many of the mRNA transcripts thought to be upregulated during the genesis of contracture formation were altered by SP inhibition, suggesting a possible dose response and reason for the lack of clinical effect.

Freeman et al. analyzed periarticular tissue from patients who developed arthrofibrosis following knee arthroplasty. They reported that the arthrofibrotic tissue was composed of dense fibroblastic regions, with limited vascularity along their outer edges. These fibrotic regions had elevated numbers of fibroblast growth factor (FGF) expressing mast cells, contained fibrocartilage and heterotopic ossification, and had positive immunostaining for lactate dehydrogenase 5, a marker of hypoxia. These authors suggested that hypoxia-associated oxidative stress may initiate mast cell proliferation and FGF secretion leading to fibroblast proliferation and tissue fibrosis.

The mast cell may thus act as the link between the acute inflammatory phase characterized by pain and swelling and the subsequent development of capsular contracture. The mast cell could thus provide an intervention target for prevention and therapeutic measures.

This role of the mast cell is supported by recent in vivo interventional studies. Red Duroc pigs show greater wound contraction than Yorkshire pigs. Ketotifen is an antiallergic and antihistaminic agent that inhibits mast cell degranulation. Ketotifen has been used in the treatment of asthma and allergic conjunctivitis. Ketotifen reduced wound contraction in red Duroc pigs to a level similar to that seen in Yorkshire pigs. Similarly, ketotifen reduced mast cell and myofibroblast numbers (by up to 65%) and the degree of flexion contractures (by 42%–52%) in immobilized fractured rabbit knees. In ketotifen-treated rabbits, protein and mRNA levels of α-SMA, TGF-β1, and collagen 1 were significantly reduced compared to untreated rabbits.

Female Sex Hormones

Female sex hormones may counteract joint fibrosis by influencing both the extracellular matrix and myofibroblasts and could potentially provide another target for medical intervention. It is well recognized that joint hypermobility is more common in females and that increased ligamentous laxity occurs in pregnancy, which would be in line with female sex hormones opposing rather than promoting fibrosis. Estrogen, progesterone, and relaxin receptors are present in the anterior cruciate ligament (ACL), and ACL laxity correlates positively with estrogen and progesterone peaks during the menstrual cycle. Estrogen and relaxin have been shown to decrease collagen synthesis while the latter may also stimulate the expression of MMPs. Sex hormone receptors are also found in myofibroblasts of normal and pathologic tissues and estrogen was shown to prevent cardiac fibrosis via activation of the myofibroblast estrogen receptor beta. Relaxin was shown to decrease myofibroblast proliferation, and downregulate the expression of α-SMA in cell cultures. Similar effects of sex hormones have been shown in in vivo fibrosis models. Relaxin therapy enhanced muscle regeneration and reduced fibrosis after skeletal muscle injury. In rat knees, contractures were created in pregnant and nonpregnant rats. Following a 2-week immobilization, there was a trend toward reduced contractures in pregnant compared to nonpregnant rats. However, this sensitivity of connective tissue to sex hormones may be more complex and modulated by injury. In rabbit knees, pregnancy increased laxity of the medial collateral ligament in uninjured rabbits.

Mechanical Factors

Differentiation of fibroblasts into myofibroblasts requires chemical stimulators but also a mechanically resistant substrate. Even in the presence of active TGF-β1, lack of stress inhibits myofibroblast differentiation. In addition, TGF-β1 upregulated α-SMA in fibroblast cultures grown on stiff collagen but not those grown on compliant collagen. These observations suggest that a vicious relation exists, whereby myofibroblasts are needed for the creation of stiff fibrotic tissue, and, in turn, stiff fibrotic tissue is needed for further myofibroblast differentiation.