Collagen Changes in Dupuytren’s Disease

Abstract

The major biochemical characteristic of Dupuytren's disease (DD) is the progressive and irreversible deposition of excess fibrous collagen. This proliferation of collagen certainly impairs normal function, but in some as yet unknown way it also provides the disease with its characteristic clinical feature, flexure of the fingers.The major questions in DD are: (1) What are the stimuli that actuate the process of collagen proliferation? (2) Can one inhibit or reverse this excess deposition of collagen? (3) What is the mechanism by which the tissue contracts? A prerequisite of any hypothesis to account for the characteristic features of DD is knowledge of the changes taking place in the collagenous structure of the aponeurosis. It cannot be assumed that all fibrotic situations are similar nor that any particular stimulating factor produces identical effects. For example, changes of collagen type follow different paths during fibrosis of the skin and kidney in scleroderma (Black et al. 1985) and during normal and hypertrophic scarring in the dermis (Bailey et al. 1975b). Any proposed stimulating factor must produce all the changes observed to occur in the collagen in DD. In contrast to most fibrotic situations, in DD one can distinguish the early and late stages of the disease. It is therefore possible to follow, at least in part, the course of the disease, and from the early stages it should be possible to distinguish between a variety of suggested stimulating factors.The lesion primarily involves the palmar aponeurosis, i.e., the collagenous fascia or fibrous sheet separating the flexor tendon from the overlying fibrofatty layer. In the early stages discrete highly cellular nodules form, but in the later stages few nodules are present and the characteristic feature is dense fibrotic bands or cords along the aponeurosis. These changes result in flexural contraction of one or more fingers towards the palm. Studies on the collagenous tissue have therefore concentrated on comparing the nodules and the contracture bands.Alterations in the Collagenous Tissue Changes in: (a) physical appearance of the fibres and their composition in terms of genetic type of collagen; (b) posttranslational modification and extracellular cross-linking and (c) the organisation of the tissues have been reported (for recent review see McFarlane et al. 1990).Biochemical Changes Studies on DD have attempted to identify and analyse at least three regions of the aponeurosis: (1) the highly cellular nodules, (2) the fibrous bands and (3) the apparently unaffected regions.Composition.There is a progressive increase in the proportion of collagen in the aponeurosis from the control, at about 60%, to the bands, at about 90% and even higher in the nodules (Bazin et al. 1980; Brinkley-Parsons et al. 1981; Hamamoto et al. 1982). The amount of neutral-salt soluble and acid soluble collagen was very small, about 0.2%, from the diseased tissue compared to virtually nothing from the control. Similarly, the amount of collagen digestible by pepsin treatment increased from 80% in the controls to almost complete solubilisation for the diseased tissue, as would be expected for immature collagen.Compositional analysis of the collagen extracted revealed a higher level of hydroxylation, increasing from five to 13 residues of hydroxylysine per 1000 residues (Brinkley-Parsons et al. 1981). This increase was accompanied by a parallel increase in the number of glycosylated hydroxylysines so that the relative proportion of glycosylated hydroxy lysines remained constant.Increased glycosylation occurred in both type I and type III collagens. The overall increase in the hydroxylation of the bands is illustrated in Fig. 1.Collagen Types.Chemical determination of the ratio of type I to type III collagen has been carried out using either pepsin digestion, which solubilised over 90% of the collagen and therefore gave a representative sample (Bailey et al. 1977; Bazin et al. 1980; Gelberman et al. 1980), or complete dissolution of the sample by cyanogen bromide in formic acid (Brinkley-Parsons et al. 1981).Similar results were obtained for ratios of types I to III. Basically there was an increase from l % - 2 % type III in the normal aponeurosis to 10%-15% in the apparently uninvolved, 10%-20% in the nodules, and 30%-40% in the fibrous bands (Fig. 1).Murrell et al. (1989) have suggested that the change in the type I to III ratio is due to a decrease in the synthesis of type I collagen. However, this proposal is based on decreased synthesis of type I collagen from fibroblasts in high density culture and may not relate to in vivo conditions. The decrease in type I necessary to account for the apparent increase in type III from 3% to 30% would be dramatic and unlikely in a fibrotic condition.These changes in collagen types determined by analysis of the tissue are analogous to those occurring in granulation tissue of dermal wounds (Bailey et al. 1975a) and in hypertrophic scars (Bailey et al. 1975b). One would expect the greatest amount of type III in the nodules, where there is a rapid proliferation of collagen, and decreasing amounts in the bands as they mature, if Dupuytren's contracture follows the pattern of normal wounds and fibrotic lesions. However, a large amount of type III would be retained over a long time period if the bands follow a similar course to those of the hypertrophic scar. Cross-link studies show that the DD bands do not mature, indicating more rapid turnover of collagen in the band. In addition, analysis of collagen from patients with long-standing Dupuytren's revealed biochemical changes similar to those in short-term disease (Brinkley-Parsons et al. 1981), indicating a failure to mature analogous to what occurs in the hypertrophic scar.Other collagen types have been detected, as in granulation tissue, but in much smaller amounts. The relative proportions of type V and type I trimer were found to double from 5% to 9% and 2% to 5%, respectively. These increases are similar to those found in hypertrophic scars (Ehrlich et al. 1982). Type VI collagen (Timpl and Engel 1987) is not detectable in normal tendon, but examination of the aponeurosis of Dupuytren's revealed the presence of type VI in the fascicular sheath. Electron microscopic examination of the nodules and bands showed typical 100 nm banded fibrils, but supporting evidence that the fibrils were type VI has not yet been provided by immunofluorescent staining.Cross-Linking.Distinct differences in the cross-link patterns were reported by Bailey and coworkers (Bailey et al. 1977; Bazin et al. 1980) and have been confirmed by others (Brinkley-Parsons et al. 1981; Gelberman et al. 1980; Hanyu et al. 1984). As anticipated for mature collagen the control tissue revealed hexosyl lysines as the major reducible components, the divalent reducible cross-links being barely detectable. In contrast, the major reducible components of the nodules and the bands were the two reducible cross-links, the aldimine dehydro-hydroxylysinonorleucine and the keto-imine, hydroxylysino-keto-norleucine (Fig. 2). The reported increased levels of lysyl oxidase (Hamamoto et al. 1982) are consistent with the higher levels of these reducible cross-links. Surprisingly the apparently unaffected parts of the aponeurosis also showed increased amounts of the reducible cross-links, although a significant level of hexosyl lysines is still present. It should be remembered that the hexosyl lysines are not cross-links but can be considered good indicators of maturity (Bailey et al. 1974). Reducible cross-links are only present in immature tissues, and hexosyl lysine in mature tissue.Hanyu et al. (1984) reported equal amounts of pyridinoline in the normal and affected aponeurosis and concluded that the cross-link was not involved in the pathogenesis of the disease. Recently we have reanalysed the tissues for the non-reducible cross-links, histidino-hydroxylysinonorleucine (HHL) and pyridinoline (Fig. 2). The levels of HHL decreased by about 50% in the bands compared to the controls, as expected for an immature tissue. In contrast, a significant increase of about threefold in the pyridinoline levels occurred in the DD bands compared to the controls (Fig. 3). This finding is consistent with the overall increase in hydroxylation and DHLNL, rather than indicating maturity of the tissue.The apparently unaffected aponeurosis shows clear signs of some newly synthesised collagen, whilst the highly active nodules contain completely new collagen. The bands are mainly newly synthesised collagen but contain some mature features similar to the control, i.e., increased hexosyl lysines, clearly indicating that some maturation of the tissue has occurred.Elastic Fibres. These fibres are a two component system consisting mainly of elastin but with a small proportion of microfibrils. Analysis of the amount of elastin was determined by the cross-links desmosine and iso-desmosine and found to be decreased by about 70% in the D D bands from a level of about 1% in the controls, consistant with increased amounts of newly synthesised collagen.Structural Changes Briefly, histological changes in D D (Hueston 1963; Larson et al. 1960; Millesi 1965; Millesi et al. 1983; Tubiana 1967) revealed two components, a highly cellular nodule and a virtually acellular scar tissue. The nodules are characterised by a network of thin collagen fibres and proteoglycans netachromatic to toluidine blue. In contrast, the fibres of the bands are more normal aponeurosis.Scanning electron microscopy has revealed similar changes (Hunter and Ogdon 1975; Legge et al. 1981). Using transmission electron microscopy it is seen that the individual collagen fibers possess the normal structure and typical axial banding pattern of 67 nm. Similarly, analysis of the fibres by both wide and low angle X-ray diffraction showed no de