Equine Veterinary Journal ISSN 0425-1644 DOI: 10.1111/evj.12278
Comparison of the blood supply to the articular-epiphyseal growth complex in horse vs. pony foals E. H. S. HENDRICKSON*, K. OLSTAD, A. NØDTVEDT, E. PAUWELS†, L. VAN HOOREBEKE† and N. I. DOLVIK Department of Companion Animal Clinical Sciences, Norwegian School of Veterinary Science, Oslo, Norway † Department of Physics and Astronomy, UGCT, Ghent University, Gent, Belgium. *Correspondence email: [email protected]; Received: 09.04.13; Accepted: 05.04.14
Summary Reasons for performing study: To increase understanding of why the prevalence of clinical/radiographic osteochondrosis (OC) dissecans is high in horses and low in ponies. Objectives: To investigate whether the clinical difference in OC occurrence between horses and ponies could partly be explained by a difference in: 1) number of patent vessels in the epiphyseal growth cartilage; 2) duration of the presence of patent cartilage canals; or 3) growth cartilage thickness at predilection sites for OC. The hypothesis was that pony foals would have fewer cartilage canals, shorter duration of blood supply and thinner growth cartilage than horse foals. Study design: Observational, cross-sectional study. Methods: Nine Standardbred foals (horse group) 1–49 days old and 11 Norwegian Fjord foals (pony group) 1–62 days old were included. A total of 15 anatomical locations in the tarsocrural and metatarsophalangeal joints were examined by one or more of the following techniques: arterial perfusion; photography of cleared specimens; microcomputed tomography; radiography; and histology. The number of cartilage canals was counted. Cartilage thickness was measured. Duration of blood supply was assessed in histological sections. Results: Of the 3 common predilection sites for OC investigated, there were significantly fewer vessels (P = 0.003) and thinner cartilage (P = 0.002) at the distal lateral trochlear ridge of the talus in the pony group. There was no difference in the duration of presence of cartilage canals between the groups. Conclusion: The hypothesis that pony foals would have fewer cartilage canals and thinner growth cartilage than horse foals was confirmed for the lateral trochlear ridge of the talus. The current results may contribute towards an explanation for the low prevalence of OC at the distal lateral trochlear ridge of the talus in pony foals. Keywords: horse; foal; metatarsophalangeal joint; tarsocrural joint; cartilage canals; osteochondrosis
Introduction Osteochondrosis (OC) is common in many species including horses, dogs, pigs and man [1,2]. Articular OC is a developmental orthopaedic disease of synovial joints that is characterised by a focal disturbance in enchondral ossification of the growth cartilage [1]. In chronic stages of the disease loose fragments of cartilage and bone can be present within the joints and defects are seen in the articular cartilage (osteochondrosis dissecans or OCD) [1]. The disease manifests at certain predilection sites specific to each joint, and are often bilaterally symmetrically distributed [3]. Osteochondrosis is common in several large and fast growing breeds of horses such as Standardbreds [4], Warmbloods [5] and Thoroughbreds [6]. In comparison, lesions reported as OC have been described in only 4 ponies and one crossbred pony [7,8]. Osteochondrosis was not found in 2 radiographic surveys of the tarsi and appendicular skeleton of Icelandic horses [9,10]. Although articular cartilage is avascular and nourished mainly by diffusion from the synovial fluid, the growth cartilage of the epiphysis is much thicker, and dependent on vessels within cartilage canals for exchange of nutrients and waste products [11,12]. As the ossification progresses, the growth cartilage becomes thinner, and the cartilage canals gradually regress by chondrification [11,13]. The remaining growth cartilage survives without blood supply. It has been suggested that this is because the diffusion distance from alternate sources of nourishment such as the synovial fluid and vessels arising from the subchondral bone is sufficient to support the cartilage [11]. The initial lesions of OC are known to arise in the same time period when the growth cartilage is dependent on vascular supply [14]. Ytrehus et al. [15] and Olstad et al. [16] performed detailed studies of the role of cartilage canals in the development of early lesions of OC in piglets and foals, respectively. They found that early lesions of OC (ischaemic chondronecrosis) were consistently located where the cartilage canal vessels traversed the ossification front to enter the distal termini of the canals. The authors pointed out that the transition from bone to cartilage exposed the canals to considerable mechanical stress, and constituted an
326
anatomical vulnerable point. They concluded that failure of cartilage canal vessels was followed by ischaemic necrosis in surrounding chondrocytes and cartilage. The aims of this study were to investigate whether the difference in occurrence of clinical OC between a horse breed with high prevalence and a pony breed with presumed low prevalence of the disease could partly be explained by: 1) a difference in number of patent cartilage canal vessels in the epiphyseal growth cartilage; 2) duration of the presence of patent cartilage canals; or 3) a difference in growth cartilage thickness at predilection sites for OC between horse and pony foals. The hypothesis was that pony foals would have fewer cartilage canals, shorter duration of blood supply and thinner growth cartilage than horse foals, making them physiologically less dependent on cartilage canal blood supply and hence possibly less susceptible to formation of the initial lesions of OC.
Materials and methods Two populations of foals allocated to horse or pony groups were used in this study. Both populations were originally bred for participation in other studies [16,17]. The horse group included 9 Standardbred foals, of which both parents except the dam of one foal were diagnosed by radiography with OCD of the tarsocrural joint. Horse foals were between 1 and 49 days of age. The pony group included 11 Norwegian Fjord foals, of which both parents were radiographically free of OC and OCD in the femoropatellar joint. The dams were also free of OC and OCD lesions of the tarsocrural and fetlock joints. Pony foals were between 1 and 62 days of age. Additional information regarding the mares and management was collected retrospectively for the purpose of the current study. Both groups of foals were born and kept at a commercial stud farm where they had 24 h turnout on pasture until euthanasia. Horse foals were born between May and October of 2004 or 2005, with 6 of the foals (66%) born in May to July. Pony foals were born between March and July of 2009, with 10 of the foals (90%) born in May to July. Horse foals were subjected to euthanasia at weekly intervals from age one to 49 days. Two of the foals were aged 14 days. Pony foals were subjected to euthanasia at ages one, 15, 17, 20, 23, Equine Veterinary Journal 47 (2015) 326–332 © 2014 EVJ Ltd
Comparison of growth cartilage blood supply between horse and pony
E. H. S. Hendrickson et al.
TABLE 1: Anatomical locations, methods and observed parameters Method/parameter observed Photography
Radiography/cartilage thickness
Histology/duration of blood supply
Bone
Loc. No.
Location
View
No. of vessels
MicroCT/cartilage thickness
Tibia
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cranial DIRT* Caudal DIRT Proximal LTR Middle LTR Distal LTR* Proximal MTR Middle MTR Distal MTR** Distal LC Distal MC Dorsal SR* Ventral SR** Plantar SR** Mid-articular Plantar
Cranial Caudal Lateral Lateral Lateral Medial Medial Medial Dorsal Dorsal Lateral Lateral Lateral
x x x x x x x x x x x x x
x
x
x
x
Talus
MtIII
P1
x x x x
CT = computed tomography; MtIII = third metatarsal bone; P1 = first phalanx; DIRT = distal intermediate ridge tibia; LTR = lateral trochlear ridge; MTR = medial trochlear ridge; LC = lateral condyle; MC = medial condyle; SR = sagittal ridge. *Common predilection site for osteochondrosis. ** Other reported predilection site for osteochondrosis.
In order to generate comparable data, the same methods as had previously been used to examine the horse group were replicated in the pony group for the purpose of the current study [16,18]. The foals were anaesthetised, and subjected to euthanasia with pentobarbital at the end of the experiment, and a detailed protocol has been published elsewhere [16]. Micronised barium was perfused into the femoral artery in order to examine arterioles within the growth cartilage. The right hind leg was perfused in one foal in the horse group; in all other foals the left hind leg was used. After completion of the perfusion, the tarsocrural and metatarsophalangeal joints were harvested, dissected free of soft tissues and disarticulated. After fixation in 4% phosphate-buffered formaldehyde for 48 h, the bones were dehydrated in increasing concentrations of alcohol, before transfer to concentrated methyl salicylate for clearing of the tissues by the modified Spalteholz technique [19]. A total of 15 anatomical locations at the distal tibia, talus, third metatarsal bone (MtIII) and first phalanx (P1) were examined. The methods used on each location are presented in Table 1, and included photography, microcomputed tomography (micro-CT), radiography and histology. Locations 1, 5 and 11 represented common predilection sites for OC. Locations 8, 12 and 13 represented less commonly reported sites for OC lesions [20]. Material, in terms of photographs, micro-CT images, radiographs, histological sections and diagnoses, was reused from the previous horse studies. All pony material, and all counts and measurements were generated for this study.
In the tarsus, cartilage thickness was evaluated from micro-CT images of sample blocks of the cranial part of the distal intermediate ridge of the tibia (DIRT) and the distal lateral trochlea ridge of the talus (LTR). The measurements were made in a CT visualisation and analysis program (VGStudio MAX)b. The numbers of image slices from the distal end of the ossification front to the distal most slice containing cartilage were counted. A measurement of cartilage thickness in mm was obtained by multiplying the number of slices with the (isotropic) voxel size. In the metatarsophalangeal joint, cartilage thickness was evaluated from radiographic images of ∼5 mm transverse bone slabs from MtIII and sagittal slabs from P1 (Table 1). Measurements were made using a picture archiving and communication system (Kodak Carestream PACS)c. For the MtIII the distal most slab was excluded, and measurements were made on the subsequent 3 proximal slabs. The maximal length from dorsal to plantar sagittal ridge was used as a measure of bone size. The cartilage thickness was measured at the dorsal and plantar aspect of the sagittal ridge (Fig 2a). For the P1 the medial most slab was excluded, and measurements were made on the subsequent 5 slabs. The width of the bone at the level of the growth plate was used as a measure of bone size and the cartilage thickness was measured at mid-joint and at the plantar aspect of the joint surface (Fig 2b). All measurements were repeated 3 times in order to correct for measurement errors, and the mean value was used for statistical analysis. Cartilage thickness was recorded both as absolute thickness, and as a ratio between cartilage thickness and the size of the bone (= relative thickness). Duration of blood supply was evaluated by histological examination. The presence or absence of patent or chondrifying cartilage canals was recorded, as was the presence or absence of areas of ischaemic chondronecrosis. This evaluation was done using the criteria of Olstad et al. (Figs 2, 3 in [13]).
Parameters observed
Data analysis
The intact, cleared bones were photographed while immersed in methyl salicylate. Photographic views are specified in Table 1. The numbers of vessels were evaluated by counting barium-filled cartilage canals visible in the growth cartilage on the photographs. An image-analysis software program (Image-Pro Plus)a was used. Each location was identified by predefined anatomical landmarks and marked with a grid. The vessels were counted using a manual tag function (Fig 1). All measurements were repeated 3 times in order to correct for measurement errors, and the mean value was used for statistical analysis.
The number of vessels and the cartilage thickness were compared between joints from pony and horse groups using separate linear regression models for each anatomical location and outcome. This allowed controlling for the confounding variables age and sex. Because several slabs were included from the MtIII and P1 from each individual, linear mixed models with random foal effects were used to evaluate the presumed effect of breed on cartilage thickness in these locations. Model fit was assessed by plotting standardised residuals in normal quantile plots. Due to the high number of comparisons, the cut-off for statistical
28, 35, 42, 48, 57 and 62 days. Three of the 6 horse dams and 6 of the 11 pony dams were primiparous. The sex distribution was 5 males and 4 females in the horse group and 7 males and 4 females in the pony group.
Data collection
Equine Veterinary Journal 47 (2015) 326–332 © 2014 EVJ Ltd
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Comparison of growth cartilage blood supply between horse and pony .
E. H. S. Hendrickson et al.
42 41
25
40 39 38 37 36 35 34 33 32 31 29 30 28 27 26
14
13 12 Fig 1: Counting of perfused cartilage canals at the distal medial trochlear ridge of the talus. The arrow is pointing towards a perfused canal. A section of the image is magnified in the lower right hand corner.
1 234 5
6
7 8
9 10
14
15
16
18 17
13 12 11 9
11
15
10
8
significance was set to Pt
Tibia Talus
1
Cranial DIRT Distal LTR1
-0.09 -1.15
0.25 0.30
MtIII
Dorsal SR1 Plantar SR2 Mid-articular Plantar
-1.16 -0.26 -0.35 -0.06
0.10 0.13 0.08 0.08
-0.36 -3.80 z -1.55 -2.02 -4.53 -0.66
0.723 0.002* P>z 0.121 0.043 0.000* 0.509
Bone
P1
Location
The coefficient denotes difference in mean cartilage thickness between groups (ponies - horses), and is calculated from linear mixed models with random foal effect. The variables age and sex are controlled for. MtIII = third metatarsal bone; P1 = first phalanx; DIRT = distal intermediate ridge tibia; LTR = lateral trochlear ridge; SR = sagittal ridge. 1Common predilection site for osteochondrosis. 2Other reported predilection site for osteochondrosis. Significant differences are marked with*. Equine Veterinary Journal 47 (2015) 326–332 © 2014 EVJ Ltd
Authorship E.H.S. Hendrickson contributed to study design, study execution, data analysis, and interpretation and the writing of the manuscript. K. Olstad contributed to the conception and design of the study, study execution, and data interpretation. A. Nødtvedt contributed to the statistical analysis. E. Pauwels and L. Van Hoorebeke contributed to collection and analysis of micro-CT data. N.I. Dolvik contributed to the conception and design of the study, and data interpretation. All authors contributed to the preparation of the manuscript, and have approved the final version of the manuscript.
Manufacturers’ addresses a
Media Cybernetics, Inc., Bethesda, Maryland, USA. Volume Graphics GmbH, Heidelberg, Germany. c Eastman Kodak Company, Rochester, New York, USA. d Stata Corp, College Station, Texas, USA. b
References 1. Ytrehus, B., Carlson, C.S. and Ekman, S. (2007) Etiology and pathogenesis of osteochondrosis. Vet. Pathol. 44, 429-448. 2. Olsson, S.E. and Reiland, S. (1978) Nature of osteochondrosis in animals – summary and conclusions with comparative aspects on osteochondritis dissecans in man. Acta Radiol. Diagn. 358, 299-305. 3. McIlwraith, C.W. (1993) Inferences from referred clinical cases of osteochondritis dissecans. Equine Vet. J. 25, 27-30. 4. Lykkjen, S., Røed, K.H. and Dolvik, N.I. (2012) Osteochondrosis and osteochondral fragments in Standardbred trotters: prevalence and relationships. Equine Vet. J. 44, 332-338. 5. van Grevenhof, E.M., Ducro, B.J., van Weeren, P.R., van Tartwijk, J.M.F.M., van den Belt, A.J. and Bijma, P. (2009) Prevalence of various radiographic manifestations of osteochondrosis and their correlations between and within joints in Dutch Warmblood horses. Equine Vet. J. 41, 11-16. 6. O’Donohue, D.D., Smith, F.H. and Strickland, K.L. (1992) The incidence of abnormal limb development in the Irish Thoroughbred from birth to 18 months. Equine Vet. J. 24, 305-309. 7. Strömberg, B. (1976) Osteochondrosis dissecans of stifle joint in horse – clinical, radiographic, and pathologic-study. J. Am. Vet. Radiol. Soc. 17, 117-124. 8. Voute, L.C., Henson, F.M., Platt, D. and Jeffcott, L.B. (2011) Osteochondrosis lesions of the lateral trochlear ridge of the distal femur in four ponies. Vet. Rec. 168, 265. doi: 10.1136/vr.c6677 9. Björnsdóttir, S., Axelsson, M., Eksell, P., Sigurdsson, H. and Carlsten, J. (2000) Radiographic and clinical survey of degenerative joint disease in the distal tarsal joints in Icelandic horses. Equine Vet. J. 32, 268-272. 10. Strand, E., Braathen, L.C., Hellsten, M.C., Huse-Olsen, L. and Björnsdóttir, S. (2007) Radiographic closure time of appendicular growth plates in the Icelandic horse. Acta Vet. Scand. 49, 19. doi: 10.1186/1751-0147-49-19 11. Carlson, C.S., Meuten, D.J. and Richardson, D.C. (1991) Ischemic necrosis of cartilage in spontaneous and experimental lesions of osteochondrosis. J. Orthop. Res. 9, 317-329. 12. Blumer, M.J., Longato, S. and Fritsch, H. (2008) Structure, formation and role of cartilage canals in the developing bone. Ann. Anat. 190, 305-315. 13. Olstad, K., Ytrehus, B., Ekman, S., Carlson, C.S. and Dolvik, N.I. (2007) Early lesions of osteochondrosis in the distal tibia of foals. J. Orthop. Res. 25, 1094-1105. 14. Carlsten, J., Sandgren, B. and Dalin, G. (1993) Development of osteochondrosis in the tarsocrural joint and osteochondral fragments in the fetlock joints of Standardbred trotters. I. A radiological survey. Equine Vet. J. 25, 42-47. 15. Ytrehus, B., Ekman, S., Carlson, C.S., Teige, J. and Reinholt, F.P. (2004) Focal changes in blood supply during normal epiphyseal growth are central in the pathogenesis of osteochondrosis in pigs. Bone 35, 1294-1306. 16. Olstad, K., Ytrehus, B., Ekman, S., Carlson, C.S. and Dolvik, N.I. (2008) Epiphyseal cartilage canal blood supply to the tarsus of foals and relationship to osteochondrosis. Equine Vet. J. 40, 30-39. 17. Olstad, K., Hendrickson, E.H.S., Carlson, C.S., Ekman, S. and Dolvik, N.I. (2013) Transection of vessels in epiphyseal cartilage canals leads to osteochondrosis and osteoshondrosis dissecans in the femoro-patellar joint of foals; a potential model of juvenile osteochondritis dissecans. Osteoarthritis Cartilage 21, 730-738.
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Comparison of growth cartilage blood supply between horse and pony
18. Olstad, K., Ytrehus, B., Ekman, S., Carlson, C.S. and Dolvik, N.I. (2009) Epiphyseal cartilage canal blood supply to the metatarsophalangeal joint of foals. Equine Vet. J. 41, 865-871. 19. Guyer, M.F. (1953) Objects of general interest. In: Animal Micrology – Practical Exercises in Zoölogical Micro-Technique, 5 edn., Ed: M.F. Guyer, University of Chicago Press, Chicago, Illinois. pp 110-113. 20. Richardson, D.W. (2011) Diagnosis and management of osteochondrosis and osseous cyst-like lesions. In: Diagnosis and Management of Lameness in the Horse, 2 edn., Eds: M.W. Ross and S.J. Dyson, Elsevier Saunders, St Louis, Missouri. pp 631-638. 21. Norwegian Horse Centre (2012) Breeding plan for the Norwegian Fjord Horse, Stiftelsen Norsk Hestesenter, Lena. 22. Bjørnstad, G., Nilsen, N.O. and Røed, K.H. (2003) Genetic relationship between Mongolian and Norwegian horses? Anim. Genet. 34, 55-58. 23. Carlson, C.S., Cullins, L.D. and Meuten, D.J. (1995) Osteochondrosis of the articular epiphyseal cartilage complex in young horses – evidence for a defect in cartilage canal blood-supply. Vet. Pathol. 32, 641-647. 24. Ytrehus, B., Haga, H.A., Mellum, C.N., Mathisen, L., Carlson, C.S., Ekman, S., Teige, J. and Reinholt, F.P. (2004) Experimental ischemia of porcine growth cartilage produces lesions of osteochondrosis. J. Orthop. Res. 22, 1201-1209. 25. Wilsman, N.J. and Van Sickle, D.C. (1972) Cartilage canals, their morphology and distribution. Anat. Rec. 173, 79-93. 26. Olstad, K., Cnudde, V., Masschaele, B., Thomassen, R. and Dolvik, N.I. (2008) Micro-computed tomography of early lesions of osteochondrosis in the tarsus of foals. Bone 43, 574-583. 27. Levene, C. (1964) The patterns of cartilage canals. J. Anat. 98, 515-538.
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28. Brunnberg, M.M., Engelke, E., Gielen, I.M., van Bree, H.J., Hoffmann, J.E., Brunnberg, L. and Waibl, H.R. (2011) Cartilage thickness of the trochlea of the talus, with emphasis on sites predisposed to osteochondrosis dissecans, in clinically normal juvenile and adult dogs. Am. J. Vet. Res. 72, 1318-1324. 29. Firth, E.C. and Greydanus, Y. (1987) Cartilage thickness measurement in foals. Res. Vet. Sci. 42, 35-46. 30. van Weeren, P.R. (2006) Etiology, diagnosis, and treatment of OC(D). Clin. Tech. Equine Pract. 5, 248-258. 31. Lykkjen, S., Olsen, H.F., Dolvik, N.I., Grøndahl, A.M., Røed, K.H. and Klemetsdal, G. (2013) Heritability estimates of tarsocrural osteochondrosis and palmar/plantar first phalanx osteochondral fragments in Standardbred trotters. Equine Vet. J. 46, 32-37. 32. Łuszczyn´ski, J., Pieszka, M. and Kosiniak-Kamysz, K. (2011) Effect of horse breed and sex on growth rate and radiographic closure time of distal radial metaphyseal growth plate. Livestock Sci. 141, 252-258.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Supplementary Item 1: Number of vessels and weight at death in horse and pony foals. Supplementary Item 2: Cartilage thickness measurements (mm) in horse and pony foals. Numbers are a mean of 3 repeated measurements.
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Equine Veterinary Journal 47 (2015) 326–332 © 2014 EVJ Ltd
Comparison of the blood supply to the articular-epiphyseal growth complex in horse vs. pony foals E. H. S. HENDRICKSON*, K. OLSTAD, A. NØDTVEDT, E. PAUWELS†, L. VAN HOOREBEKE† and N. I. DOLVIK Department of Companion Animal Clinical Sciences, Norwegian School of Veterinary Science, Oslo, Norway † Department of Physics and Astronomy, UGCT, Ghent University, Gent, Belgium. *Correspondence email: [email protected]; Received: 09.04.13; Accepted: 05.04.14
Summary Reasons for performing study: To increase understanding of why the prevalence of clinical/radiographic osteochondrosis (OC) dissecans is high in horses and low in ponies. Objectives: To investigate whether the clinical difference in OC occurrence between horses and ponies could partly be explained by a difference in: 1) number of patent vessels in the epiphyseal growth cartilage; 2) duration of the presence of patent cartilage canals; or 3) growth cartilage thickness at predilection sites for OC. The hypothesis was that pony foals would have fewer cartilage canals, shorter duration of blood supply and thinner growth cartilage than horse foals. Study design: Observational, cross-sectional study. Methods: Nine Standardbred foals (horse group) 1–49 days old and 11 Norwegian Fjord foals (pony group) 1–62 days old were included. A total of 15 anatomical locations in the tarsocrural and metatarsophalangeal joints were examined by one or more of the following techniques: arterial perfusion; photography of cleared specimens; microcomputed tomography; radiography; and histology. The number of cartilage canals was counted. Cartilage thickness was measured. Duration of blood supply was assessed in histological sections. Results: Of the 3 common predilection sites for OC investigated, there were significantly fewer vessels (P = 0.003) and thinner cartilage (P = 0.002) at the distal lateral trochlear ridge of the talus in the pony group. There was no difference in the duration of presence of cartilage canals between the groups. Conclusion: The hypothesis that pony foals would have fewer cartilage canals and thinner growth cartilage than horse foals was confirmed for the lateral trochlear ridge of the talus. The current results may contribute towards an explanation for the low prevalence of OC at the distal lateral trochlear ridge of the talus in pony foals. Keywords: horse; foal; metatarsophalangeal joint; tarsocrural joint; cartilage canals; osteochondrosis
Introduction Osteochondrosis (OC) is common in many species including horses, dogs, pigs and man [1,2]. Articular OC is a developmental orthopaedic disease of synovial joints that is characterised by a focal disturbance in enchondral ossification of the growth cartilage [1]. In chronic stages of the disease loose fragments of cartilage and bone can be present within the joints and defects are seen in the articular cartilage (osteochondrosis dissecans or OCD) [1]. The disease manifests at certain predilection sites specific to each joint, and are often bilaterally symmetrically distributed [3]. Osteochondrosis is common in several large and fast growing breeds of horses such as Standardbreds [4], Warmbloods [5] and Thoroughbreds [6]. In comparison, lesions reported as OC have been described in only 4 ponies and one crossbred pony [7,8]. Osteochondrosis was not found in 2 radiographic surveys of the tarsi and appendicular skeleton of Icelandic horses [9,10]. Although articular cartilage is avascular and nourished mainly by diffusion from the synovial fluid, the growth cartilage of the epiphysis is much thicker, and dependent on vessels within cartilage canals for exchange of nutrients and waste products [11,12]. As the ossification progresses, the growth cartilage becomes thinner, and the cartilage canals gradually regress by chondrification [11,13]. The remaining growth cartilage survives without blood supply. It has been suggested that this is because the diffusion distance from alternate sources of nourishment such as the synovial fluid and vessels arising from the subchondral bone is sufficient to support the cartilage [11]. The initial lesions of OC are known to arise in the same time period when the growth cartilage is dependent on vascular supply [14]. Ytrehus et al. [15] and Olstad et al. [16] performed detailed studies of the role of cartilage canals in the development of early lesions of OC in piglets and foals, respectively. They found that early lesions of OC (ischaemic chondronecrosis) were consistently located where the cartilage canal vessels traversed the ossification front to enter the distal termini of the canals. The authors pointed out that the transition from bone to cartilage exposed the canals to considerable mechanical stress, and constituted an
326
anatomical vulnerable point. They concluded that failure of cartilage canal vessels was followed by ischaemic necrosis in surrounding chondrocytes and cartilage. The aims of this study were to investigate whether the difference in occurrence of clinical OC between a horse breed with high prevalence and a pony breed with presumed low prevalence of the disease could partly be explained by: 1) a difference in number of patent cartilage canal vessels in the epiphyseal growth cartilage; 2) duration of the presence of patent cartilage canals; or 3) a difference in growth cartilage thickness at predilection sites for OC between horse and pony foals. The hypothesis was that pony foals would have fewer cartilage canals, shorter duration of blood supply and thinner growth cartilage than horse foals, making them physiologically less dependent on cartilage canal blood supply and hence possibly less susceptible to formation of the initial lesions of OC.
Materials and methods Two populations of foals allocated to horse or pony groups were used in this study. Both populations were originally bred for participation in other studies [16,17]. The horse group included 9 Standardbred foals, of which both parents except the dam of one foal were diagnosed by radiography with OCD of the tarsocrural joint. Horse foals were between 1 and 49 days of age. The pony group included 11 Norwegian Fjord foals, of which both parents were radiographically free of OC and OCD in the femoropatellar joint. The dams were also free of OC and OCD lesions of the tarsocrural and fetlock joints. Pony foals were between 1 and 62 days of age. Additional information regarding the mares and management was collected retrospectively for the purpose of the current study. Both groups of foals were born and kept at a commercial stud farm where they had 24 h turnout on pasture until euthanasia. Horse foals were born between May and October of 2004 or 2005, with 6 of the foals (66%) born in May to July. Pony foals were born between March and July of 2009, with 10 of the foals (90%) born in May to July. Horse foals were subjected to euthanasia at weekly intervals from age one to 49 days. Two of the foals were aged 14 days. Pony foals were subjected to euthanasia at ages one, 15, 17, 20, 23, Equine Veterinary Journal 47 (2015) 326–332 © 2014 EVJ Ltd
Comparison of growth cartilage blood supply between horse and pony
E. H. S. Hendrickson et al.
TABLE 1: Anatomical locations, methods and observed parameters Method/parameter observed Photography
Radiography/cartilage thickness
Histology/duration of blood supply
Bone
Loc. No.
Location
View
No. of vessels
MicroCT/cartilage thickness
Tibia
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cranial DIRT* Caudal DIRT Proximal LTR Middle LTR Distal LTR* Proximal MTR Middle MTR Distal MTR** Distal LC Distal MC Dorsal SR* Ventral SR** Plantar SR** Mid-articular Plantar
Cranial Caudal Lateral Lateral Lateral Medial Medial Medial Dorsal Dorsal Lateral Lateral Lateral
x x x x x x x x x x x x x
x
x
x
x
Talus
MtIII
P1
x x x x
CT = computed tomography; MtIII = third metatarsal bone; P1 = first phalanx; DIRT = distal intermediate ridge tibia; LTR = lateral trochlear ridge; MTR = medial trochlear ridge; LC = lateral condyle; MC = medial condyle; SR = sagittal ridge. *Common predilection site for osteochondrosis. ** Other reported predilection site for osteochondrosis.
In order to generate comparable data, the same methods as had previously been used to examine the horse group were replicated in the pony group for the purpose of the current study [16,18]. The foals were anaesthetised, and subjected to euthanasia with pentobarbital at the end of the experiment, and a detailed protocol has been published elsewhere [16]. Micronised barium was perfused into the femoral artery in order to examine arterioles within the growth cartilage. The right hind leg was perfused in one foal in the horse group; in all other foals the left hind leg was used. After completion of the perfusion, the tarsocrural and metatarsophalangeal joints were harvested, dissected free of soft tissues and disarticulated. After fixation in 4% phosphate-buffered formaldehyde for 48 h, the bones were dehydrated in increasing concentrations of alcohol, before transfer to concentrated methyl salicylate for clearing of the tissues by the modified Spalteholz technique [19]. A total of 15 anatomical locations at the distal tibia, talus, third metatarsal bone (MtIII) and first phalanx (P1) were examined. The methods used on each location are presented in Table 1, and included photography, microcomputed tomography (micro-CT), radiography and histology. Locations 1, 5 and 11 represented common predilection sites for OC. Locations 8, 12 and 13 represented less commonly reported sites for OC lesions [20]. Material, in terms of photographs, micro-CT images, radiographs, histological sections and diagnoses, was reused from the previous horse studies. All pony material, and all counts and measurements were generated for this study.
In the tarsus, cartilage thickness was evaluated from micro-CT images of sample blocks of the cranial part of the distal intermediate ridge of the tibia (DIRT) and the distal lateral trochlea ridge of the talus (LTR). The measurements were made in a CT visualisation and analysis program (VGStudio MAX)b. The numbers of image slices from the distal end of the ossification front to the distal most slice containing cartilage were counted. A measurement of cartilage thickness in mm was obtained by multiplying the number of slices with the (isotropic) voxel size. In the metatarsophalangeal joint, cartilage thickness was evaluated from radiographic images of ∼5 mm transverse bone slabs from MtIII and sagittal slabs from P1 (Table 1). Measurements were made using a picture archiving and communication system (Kodak Carestream PACS)c. For the MtIII the distal most slab was excluded, and measurements were made on the subsequent 3 proximal slabs. The maximal length from dorsal to plantar sagittal ridge was used as a measure of bone size. The cartilage thickness was measured at the dorsal and plantar aspect of the sagittal ridge (Fig 2a). For the P1 the medial most slab was excluded, and measurements were made on the subsequent 5 slabs. The width of the bone at the level of the growth plate was used as a measure of bone size and the cartilage thickness was measured at mid-joint and at the plantar aspect of the joint surface (Fig 2b). All measurements were repeated 3 times in order to correct for measurement errors, and the mean value was used for statistical analysis. Cartilage thickness was recorded both as absolute thickness, and as a ratio between cartilage thickness and the size of the bone (= relative thickness). Duration of blood supply was evaluated by histological examination. The presence or absence of patent or chondrifying cartilage canals was recorded, as was the presence or absence of areas of ischaemic chondronecrosis. This evaluation was done using the criteria of Olstad et al. (Figs 2, 3 in [13]).
Parameters observed
Data analysis
The intact, cleared bones were photographed while immersed in methyl salicylate. Photographic views are specified in Table 1. The numbers of vessels were evaluated by counting barium-filled cartilage canals visible in the growth cartilage on the photographs. An image-analysis software program (Image-Pro Plus)a was used. Each location was identified by predefined anatomical landmarks and marked with a grid. The vessels were counted using a manual tag function (Fig 1). All measurements were repeated 3 times in order to correct for measurement errors, and the mean value was used for statistical analysis.
The number of vessels and the cartilage thickness were compared between joints from pony and horse groups using separate linear regression models for each anatomical location and outcome. This allowed controlling for the confounding variables age and sex. Because several slabs were included from the MtIII and P1 from each individual, linear mixed models with random foal effects were used to evaluate the presumed effect of breed on cartilage thickness in these locations. Model fit was assessed by plotting standardised residuals in normal quantile plots. Due to the high number of comparisons, the cut-off for statistical
28, 35, 42, 48, 57 and 62 days. Three of the 6 horse dams and 6 of the 11 pony dams were primiparous. The sex distribution was 5 males and 4 females in the horse group and 7 males and 4 females in the pony group.
Data collection
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Comparison of growth cartilage blood supply between horse and pony .
E. H. S. Hendrickson et al.
42 41
25
40 39 38 37 36 35 34 33 32 31 29 30 28 27 26
14
13 12 Fig 1: Counting of perfused cartilage canals at the distal medial trochlear ridge of the talus. The arrow is pointing towards a perfused canal. A section of the image is magnified in the lower right hand corner.
1 234 5
6
7 8
9 10
14
15
16
18 17
13 12 11 9
11
15
10
8
significance was set to Pt
Tibia Talus
1
Cranial DIRT Distal LTR1
-0.09 -1.15
0.25 0.30
MtIII
Dorsal SR1 Plantar SR2 Mid-articular Plantar
-1.16 -0.26 -0.35 -0.06
0.10 0.13 0.08 0.08
-0.36 -3.80 z -1.55 -2.02 -4.53 -0.66
0.723 0.002* P>z 0.121 0.043 0.000* 0.509
Bone
P1
Location
The coefficient denotes difference in mean cartilage thickness between groups (ponies - horses), and is calculated from linear mixed models with random foal effect. The variables age and sex are controlled for. MtIII = third metatarsal bone; P1 = first phalanx; DIRT = distal intermediate ridge tibia; LTR = lateral trochlear ridge; SR = sagittal ridge. 1Common predilection site for osteochondrosis. 2Other reported predilection site for osteochondrosis. Significant differences are marked with*. Equine Veterinary Journal 47 (2015) 326–332 © 2014 EVJ Ltd
Authorship E.H.S. Hendrickson contributed to study design, study execution, data analysis, and interpretation and the writing of the manuscript. K. Olstad contributed to the conception and design of the study, study execution, and data interpretation. A. Nødtvedt contributed to the statistical analysis. E. Pauwels and L. Van Hoorebeke contributed to collection and analysis of micro-CT data. N.I. Dolvik contributed to the conception and design of the study, and data interpretation. All authors contributed to the preparation of the manuscript, and have approved the final version of the manuscript.
Manufacturers’ addresses a
Media Cybernetics, Inc., Bethesda, Maryland, USA. Volume Graphics GmbH, Heidelberg, Germany. c Eastman Kodak Company, Rochester, New York, USA. d Stata Corp, College Station, Texas, USA. b
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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Supplementary Item 1: Number of vessels and weight at death in horse and pony foals. Supplementary Item 2: Cartilage thickness measurements (mm) in horse and pony foals. Numbers are a mean of 3 repeated measurements.
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