Histological and Radiographic Evaluation of Equine Bone Structure after Implantation of Castor Oil Polymer.

OBJECTIVE: The main purpose of this study was to evaluate the characteristics of a vegetal polymeric biomaterial intended for bone substitution in horses and to investigate the responses of the equine third metacarpal bone to biomaterial implantation. MATERIALS AND METHODS: Six horses were submitted to osteotomy on the dorsal aspect of the left and right third metacarpal bones; one bone defect was randomly selected for treatment with biopolymer, while the other was left untreated and served as a control. Bone density was monitored radiographically after surgery and bone biopsy fragments were collected at the end of the 120-day follow-up period. Biopsy fragments were analysed using light and scanning electron microscopy. RESULTS: Mean bone density values (mmAL) were greater in control defects (16.33 ± 1.6) than in polymer-treated defects (14.17 ± 1.7) at 120 days (p = 0.027). Light microscopy revealed greater percentages of new bone formation in control defects (50.15 ± 14.8) than in polymer treated defects (26.94 ± 12.1) at 120 days (p < 0.0001). Scanning electron microscopy analysis suggested a similar quality of pre-existing bone and new bone formed in the presence of biomaterial. CLINICAL SIGNIFICANCE: The absence of adverse reactions supports biomaterial biocompatibility and osteoconducting capacity and suggests the castor oil polymer is a suitable bone substitute for the treatment of bone defects in horses.

Dynamic imaging of the growth plate cartilage reveals multiple contributors to skeletal morphogenesis.

The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

Thoroughbred horses in race training have lower levels of subchondral bone remodelling in highly loaded regions of the distal metacarpus compared to horses resting from training.

Bone is repaired by remodelling, a process influenced by its loading environment. The aim of this study was to investigate the effect of a change in loading environment on bone remodelling by quantifying bone resorption and formation activity in the metacarpal subchondral bone in Thoroughbred racehorses. Sections of the palmar metacarpal condyles of horses in race training (n = 24) or resting from training (n = 24) were examined with light microscopy and back scattered scanning electron microscopy (BSEM). Bone area fraction, osteoid perimeter and eroded bone surface were measured within two regions of interest: (1) the lateral parasagittal groove (PS); (2) the lateral condylar subchondral bone (LC). BSEM variables were analysed for the effect of group, region and interaction with time since change in work status. The means ± SE are reported. For both regions of interest in the training compared to the resting group, eroded bone surface was lower (PS: 0.39 ± 0.06 vs. 0.65 ± 0.07 per mm, P = 0.010; LC: 0.24 ± 0.04 vs. 0.85 ± 0.10 per mm, P < 0.001) and in the parasagittal groove osteoid perimeter was higher (0.23 ± 0.04% vs. 0.12 ± 0.02%). Lower porosity was observed in the subchondral bone, reflected by a higher bone area fraction in the LC of the training group (90.8 ± 0.6%) compared to the resting group (85.3 ± 1.4%, P = 0.0010). Race training was associated with less bone resorption and more bone formation in the subchondral bone of highly loaded areas of the distal metacarpus limiting the replacement of fatigued bone. Periods of reduced intensity loading are important for facilitating subchondral bone repair in Thoroughbred racehorses.

Immunolocalisation of fibrillin microfibrils in the calf metacarpal and vertebral growth plate.

Overgrowth of limbs and spinal deformities are typical clinical manifestations of Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), caused by mutations of the genes encoding fibrillin-1 (FBN1) and fibrillin-2 (FBN2), respectively. FBN1 mutations are also associated with acromicric (AD) and geleophysic dysplasias (GD), and with Weill-Marchesani syndrome (WMS), which is characterised by short stature. The mechanisms leading to such abnormal skeletal growth and the involvement of the fibrillins are not understood. Postnatal longitudinal bone growth mainly occurs in the epiphyseal growth plate. Here we investigated the organisation of fibrillin microfibrils in the growth plate of the long bone and vertebra immunohistochemically. Fibrillin-1 was dual-immunostained with elastin, with fibrillin-2 or with collagen X. We report that fibrillin microfibrils are distributed throughout all regions of the growth plate, and that fibrillin-1 and fibrillin-2 were differentially organised. Fibrillin-1 was more abundant in the extracellular matrix of the resting and proliferative zones of the growth plate than in the hypertrophic zone. More fibrillin-2 was found in the calcified region than in the other regions. No elastin fibres were observed in either the proliferative or hypertrophic zones. This study indicates that, as fibrillin microfibrils are involved in growth factor binding and may play a mechanical role, they could be directly involved in regulating bone growth. Hence, mutations of the fibrillins could affect their functional role in growth and lead to the growth disorders seen in patients with MFS, CCA, AD, GD and WMS.

Exercise-induced inhibition of remodelling is focally offset with fatigue fracture in racehorses.

UNLABELLED: Bone remodelling is inhibited by high repetitive loading. However, in subchondral bone of racehorses in training, eroded surface doubled in association with fatigue fracture and there was greater surrounding trabecular bone volume suggesting trabecular modelling unloads the bone focally, allowing damage repair by remodelling. INTRODUCTION: Remodelling replaces damaged bone with new bone but is suppressed during high magnitude repetitive loading when damage is most likely. However, in cortical bone of racehorses, at sites of fatigue fracture, focal porosity, consistent with remodelling, is observed in proportion to the extent of surrounding callus. Focal areas of porosity are also observed at sites of fatigue damage in subchondral bone. We hypothesised that fatigued subchondral bone, like damaged cortical bone, is remodelled focally in proportion to the modelling of surrounding trabecular bone. METHODS: Eroded and mineralizing surfaces and bone area were measured using backscattered scanning electron microscopy of post-mortem specimens of the distal third metacarpal bone in 11 racehorses with condylar fractures (cases) and eight racehorses in training without fractures (controls). RESULTS: Cases had a two-fold greater eroded surface per unit area at the fracture site than controls (0.81 ± 0.10 vs. 0.40 ± 0.12 mm(-1), P = 0.021) but not at an adjacent site (0.22 ± 0.09 vs. 0.30 ± 0.11 mm(-1), P = 0.59). Area fraction of surrounding trabecular bone was higher in cases than controls (81 ± 2 vs. 72 ± 2 %, P = 0.0020) and the eroded surface at the fracture site correlated with the surrounding trabecular area (adjusted R (2) = 0.63, P = 0.0010). CONCLUSION: In conclusion, exercise-induced inhibition of remodelling is offset at sites of fatigue fracture. Modelling of trabecular bone may contribute to unloading these regions, allowing repair by remodelling.

Osteonal lamellae elementary units: lamellar microstructure, curvature and mechanical properties.

The mechanical and structural properties of the sublayers of osteonal lamellae were studied. Young's modulus (E) of adjacent individual lamellae was measured by nanoindentation of parallel slices every 1-3 µm, in planes parallel and perpendicular to the osteon axis (OA). In planes parallel to the OA, the modulus of a lamella could vary significantly between sequential slices. Significant modulus variations were also sometimes found on opposing sides of the osteonal canal for the same lamella. These results are rationalized by considerations involving the microstructural organization of the collagen fibrils in the lamellae. Scanning electron microscope imaging of freeze fractured surfaces revealed that the substructure of a single lamella can vary significantly on the opposing sides of the osteonal axis. Using a serial surface view method, parallel planes were exposed every 8-10 nm using a dual-beam microscope. Analysis of the orientations of fibrils revealed that the structure is rotated plywood like, consisting of unidirectional sublayers of fibrils of several orientations, with occasional randomly oriented sublayers. The dependence of the measured mechanical properties of the lamellae on the indentation location may be explained by the observed structure, as well as by the curvature of the osteonal lamellae through simple geometrical-structural considerations. Mechanical advantages arising from the curved laminate structure are discussed.

High resolution microscopic survey of third metacarpal articular calcified cartilage and subchondral bone in the juvenile horse: possible implications in chondro-osseous disease.

The aim was to survey articular calcified cartilage (ACC) and subchondral bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of young horses with minimal or no signs of musculo-skeletal abnormality. Back-scattered electron scanning electron microscopy (BSE SEM) was conducted on polymethyl methacrylate-embedded mediolateral slices and macerated wedges of the right distal Mc3 from seven each of trained and untrained 2-year-old Thoroughbred horses. Furrows or grooves visible to the naked eye in the mineralizing front (MF) of ACC are the commonest "lesion" and are most common in the palmar portions of the medial and lateral condylar grooves. Cracks running predominantly in the parasagittal plane that infill with hypercalcified matrix are found in the same domain. Common to all these defects are deficiencies or absence of the ACC MF. Other anomalies include local excrescences or depressed areas of the MF. More important condylar lesions show displaced fragmented hypermineralized ACC with underlying excess resorption in the bone domain, leaving a thin ACC layer with cavernous space beneath it. The fragments may dislodge and displace to the joint space. Obvious although small lesions are present in horses that have undertaken little or no training. The nature and sites of the lesions indicate that they are possibly the earliest morphological evidence of changes that may lead to specific joint abnormalities. The lesions appear unlikely to be solely due to functional traumatic forces, and developmental influences are likely to be important in their initiation.