Regional differences in biochemical, biomechanical and histomorphological characteristics of the equine suspensory ligament M. V. SOUZA †‡ *, P. R. VAN WEEREN † , H. T. M. VAN SCHIE †§ and C. H. A. VAN DE LEST †¶ † Department of Equine Sciences, Faculty of Veterinary Medicine; ¶ Department of Veterinary Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Yalelaan, Utrecht; § Department of Orthopaedics, Erasmus MC University Medical Centre, Rotterdam, The Netherlands; and ‡ Departamento de Veterinária, Universidade Federal de Viçosa, Campus Universitário, Minas Gerais, Brazil. Keywords: horse; musculus interosseus medius; biochemistry; biomechanics; microscopy Summary Reasons for performing study: Desmopathies of the suspensory ligament are relatively frequent and vary in prevalence over different anatomical regions. This variation may be because of regional differences in tissue characteristics. Objective: To characterise different regions of healthy forelimb suspensory ligaments (SLs) by means of biochemical, biomechanical and histomorphological techniques. Hypothesis: There is substantial topographical heterogeneity in the SL with respect to structural, compositional and functional characteristics. Methods: SLs were harvested from 38 limbs and used for biochemical (n = 20), biomechanical (n = 14) and histomorphological (n = 4) evaluation. Sulphated glycosaminoglycan (S-GAG), DNA and collagen content, degree of lysyl hydroxylation and numbers of enzymatic and nonenzymatic cross-links were determined in 7 regions of the SL: lateral and medial part of the origin (OM, OL), mid-body (MB), axial and abaxial parts of the lateral and medial branches (ILAX, ILAB, IMAX and IMAB, respectively). Passive resistance to tensile loading was measured in 5 regions of the SL (all except OL and OM). Results: DNA content was lower in OL and OM than in all other parts. GAG content was also lower in OL and OM and highest in ILAB and IMAB. Collagen content was lower in OL/OM and highest in ILAX/IMAX. Pentosidine levels were highest in OL and significantly lower in the lateral insertion (ILAX/ ILAB). There were no differences in hydroxylysylpyridinoline (HP) cross-links or lysyl hydroxylation. Stiffness (P<0.01) and modulus of elasticity (P<0.01) were substantially higher in the MB region than in all other regions except for IMAB. Strain at failure was lower in the MB region (P<0.0001), resulting in a comparable force at rupture as in the other regions. Conclusions: Matrix composition differs to a relatively limited extent between different regions of the SL. The mid-body of the ligament is stiffer than the branches, which have similar properties and relevance and mechanical differences between mid-body and branches/origin may explain some use-related differences in the prevalence of lesions. Introduction Lesions of the triad of tendonous and ligamentous structures located at the palmar or plantar side of the equine limb that are commonly denominated as the ‘flexor structures’ are frequent in performance horses. Of these structures the superficial digital flexor tendon (SDFT) is the most frequently affected (especially in racehorses), but the interosseous muscle, commonly known as suspensory ligament (SL) also is a frequently injured structure in both fore- and hindlimbs of several breeds of horses (Dyson 1994; Gibson and Steel 2002; Crowe et al. 2004; Singer et al. 2008; Tóth et al. 2008; Waselau et al. 2008). Suspensory ligament injury is encountered in racehorses (Wilson et al. 1991; Patterson-Kane et al. 1998), showjumping, dressage, eventing (Dyson 1991; Marks 1999; Crowe et al. 2004), endurance (Dyson 1998), polo (Wollenman 1999), as well as in cutting and reining (Jackman 2001). The character of suspensory ligament injuries varies and is related to the equestrian discipline involved. Forelimb high suspensory disease is primarily an insertion desmopathy with or without significant desmitis (Marks 1999). Hindlimb high suspensory disease is associated with a straight hock (Dyson 1998), and Grand Prix dressage horses with this disease may have to be retired. Front or hind suspensory body and branches lesions are the most common cause of athletic retirement in jumpers (Marks 1999). Pigeon-toed polo horses may develop lateral branch suspensory desmitis (Wollenman 1999). In general, body lesions are recognised less frequently and are often a sequel to a previous branch injury (Dyson 1998). In a recent study it was shown that 21% of horses intending to compete in Course Complète Internationale (CCI) competitions did not start due to injury. Forty-three percent of these injuries involved soft tissue structures with injuries to the SDFT and the SL each accounting for 33% (Singer et al. 2008); this means that SL injury may impede 3% of all horses to compete. Tendons and ligaments are dense fibrous connective tissues characterised by the occurrence of sparse fibroblasts embedded in a highly organised extracellular matrix (ECM). The complex structural hierarchy of ligaments and tendons permits elastic (reversible) and eventually plastic (not fully reversible) deformation under high tensile loads, which is essential for their energy storing function, but makes them vulnerable for *Corresponding author email: msouza@ufv.br [Paper received for publication 26.08.09; Accepted: 20.12.09] EQUINE VETERINARY JOURNAL 611 Equine vet. J. (2010) 42 (7) 611-620 doi: 10.1111/j.2042-3306.2010.0089.x © 2010 EVJ Ltd