Biochemical and Histological Differences between Longitudinal and Vertical Fibres of Dupuytren's Palmar Aponeurosis and Innovative Clinical Implications

Abstract

Dupuytren's disease, a chronic and progressive fibroproliferative lesion of the hand, which affects the palmar fascia, has a recurrence rate after selective aponeurotomy of 20-40% at 5 years. This study focused, for the first time, on the microanatomical and histopathological characteristics of the longitudinal and vertical fibres (usually spared during surgery) in the aponeurosis with Dupuytren's disease, in different stages of the Tubiana's classification. Twelve human samples were collected and analysed by immunostaining, Total Collagen Assay, ELISA Immunoassay, and immunoblotting for the Von Willebrand factor, α-Sma, D2-40, CD-68, Total Collagen, Collagen-I and III, IL1β, TNF-α to analyse the blood and lymphatic vascularization, the amount and distribution of collagen, and the inflammation. The results show a progressive increase in the arterial vascularization in the vertical fibres (from 8.8/mm² in the early stage to 21.4/mm² in stage 3/4), and a parallel progressive decrease in the lymphatic drainage (from 6.2/mm² to 2.8/mm²), correlated with a local inflammatory context (increase in IL-1β and TNF-α until the stage 2) in both the longitudinal and vertical fibres. The acute inflammation after stage 2 decreased, in favour of a fibrotic action, with the clear synthesis of new collagen (up to ~83 µg/mg), especially Collagen-I. These results clearly demonstrate the involvement of the septa of Legueu and Juvara in the disease pathology and the modifications with the disease's progression. A greater understanding of the pathology becomes fundamental for staging and the adequate therapeutic timing, to obtain the best morpho-functional result and the lowest risk of complications.

Keywords: Dupuytren’s disease; collagen; fibrosis; inflammation; palmar fascia.

Figure 1

Haematoxylin-eosin staining of a section of palmar fascia from Dupuytren’s (A), in which are evident the longitudinal (L) and vertical (V) fibres. (B): Haematoxylin-eosin staining of the longitudinal fibres. (C): Haematoxylin-eosin staining of the vertical fibres. In (B,C) are shown areas of dense connective tissue (D) and loose connective tissue (L). (D): Macroscopic histological image of the hand section from autopsy, stained by haematoxylin-eosin. Long: longitudinal fibres of Wood Jones. Trasv: transversal fibres of Skoog. Vert: vertical septa of Legueu and Juvara. Scale bars: (A) = 2 mm; (B,C) = 200 µm; (D) = 500 µm.

Figure 2

Anti-Von Willebrand Factor on vertical (A) and longitudinal (B) fibres, with positive endothelial cells. Scale bars: (A,B) = 50 µm. (C): Mean density of vessels obtained by anti-Von Willebrand Factor antibody stain, in longitudinal and vertical fibres of palmar aponeurosis of Dupuytren’s disease (mean values/mm² ± standard deviation).

Figure 3

Anti-α-SMA on vertical (A,B) and longitudinal (C,D) fibres at stage 2 of the pathology. Arrows indicate arteries positive to anti-α-SMA antibody. Scale bars: (A,C) = 200 µm; (B,D) = 50 µm.

Figure 4

Mean density of arterial vessels and linear trend lines obtained by anti-α-SMA antibody stain in the longitudinal and vertical fibres of palmar aponeurosis of Dupuytren’s disease at early stage, stage 1, stage 2, and stage 3–4.

Figure 5

Anti-D2-40 on the vertical (A) and longitudinal (B) fibres. Arrows indicate positive lymphatic vessels. Scale bars: (A,B) = 50 µm.

Figure 6

Mean density of lymphatic vessels and linear trend lines obtained by anti-D2-40 antibody in longitudinal and vertical fibres of palmar aponeurosis of Dupuytren’s disease at early stage, stage 1, stage 2, and stage 3–4.

Figure 7

Anti-Collagen-I (A,B) and anti-Collagen-III (C,D) in vertical (A–C) and longitudinal (B–D) fibres of palmar aponeurosis at stage 2 of Dupuytren’s disease. Scale bars: 200 µm.

Figure 8

Total collagen amount (µg/mg if tissue) in the longitudinal and vertical fibres in the different stages of the pathology. Statistical differences between the four considered stages were tested by one-way analysis of variance, followed by Tukey’s test for multiple comparisons: * p < 0.05; ** p < 0.01.

Figure 9

Normalized protein values and linear trend lines of Collagen I (A) and Collagen III (B) obtained by immunoblotting in the longitudinal and vertical fibres in the various stages of the Dupuytren’s disease (from early stage up to stage 3/4). (C) shows the chemiluminescent signal for Collagen I and Collagen III and one membrane stained with Ponceau S. M: marker; L: longitudinal fibres; V: vertical fibres.

Figure 10

Anti-CD68 on vertical (A) and longitudinal (B) fibres at early stage of Dupuytren’s disease. (C) shows the negative control with the omission of the primary antibody. (D) is the immunostaining on longitudinal fibres at stage 2 of the pathology. Arrows indicate monocytes; arrowheads indicate mast cells. Scale bars: (A,B): 50 µm, (C,D): 100 µm.

Figure 11

Chemiluminescent signal (A). (B) normalized protein values with relative trend lines of IL-1β obtained by immunoblotting in the longitudinal and vertical fibres at the various stages of the Dupuytren’s disease. M: marker; L: longitudinal fibres; V: vertical fibres.

Figure 12

Levels of TNF-α (pg per mg of total proteins) obtained by ELISA immunoassay in tissue lysates of longitudinal and vertical fibres of the palmar aponeurosis from the early to advanced stages of Dupuytren’s disease. Statistical differences between the four considered stages were tested by one-way analysis of variance, followed by Tukey’s test for multiple comparisons: * p < 0.05; ** p < 0.01.

Figure 13

Sample of palmar aponeurosis of a patient affected by Dupuytren disease, observed in the dorsal–palmar direction: the deep surface of the longitudinal fibres is evident, with a physiological layer of loose connective tissue and adipose tissue adherent to the fibres. The longitudinal fibres continue towards the digital cords, partly visible in this picture, and towards the transverse fibres of Skoog and the vertical septa joined at the superficial origin and were sectioned at their deep end.